I investigated highway mortality and the attempted crossings of turtles and other herpetofauna from 2000-2003 on a 1.2-km-section of a four-lane highway crossing Lake Jackson, Florida. U.S. Highway 27 was built directly through the northwest arm of Lake Jackson, separating a 21-ha portion of the lake to the west (now known as “Little Lake Jackson”). U.S. Highway 27 is a virtually impassable barrier to wildlife (21,500 vehicles per day) and prevents normal movements, dispersal, and migration of most species both during non-drought periods and periodic natural drydowns (on average every 12 yrs). During periods of drought, Little Lake Jackson maintains water and is the destination for large numbers of migrating turtles and other wildlife that leave the drying lake until it refi lls. The objectives of this study were to: (1) determine the level of road mortality and attempted crossings of turtles and other herpetofauna, (2) examine the potential effects of road mortality on turtle demography including sex ratios and female population size, and (3) design and evaluate the effectiveness of a temporary drift fence-culvert system to both reduce road mortality and facilitate migration. Two drift fences (885 and 600m) were constructed to divert animals away from the north and southbound lanes and direct them into an existing 3.5-m-diameter culvert. Monitoring of road kills and attempted crossings consisted of multiple daily surveys for 43 months (5558 h) including a pre-fence (40 d) and post-fence (1274 d) survey during a drought migration and non-drought conditions. A total of 10,180 reptiles and amphibians of 44 species were found either road killed or alive behind drift fences: 8,833 turtles, 825 frogs, 344 snakes, 145 lizards, 31 alligators, and 2 salamanders. Diversity among taxonomic groups included 10 species of turtles, 15 species of snakes, 10 species of anurans, 6 species of lizards, 2 salamander species, and 1 crocodilian. Drift fences combined with intensive monitoring greatly reduced turtle road kills and facilitated the use of an under-highway culvert. Pre-fence turtle mortality (9.7/km/day) was signifi cantly greater than post-fence mortality (0.08/km/day), and only 84 of 8,466 turtles climbed or penetrated the temporary fences. Pre-fence data provided strong evidence that turtles cannot successfully cross all four lanes of U.S. Highway 27, with 95 percent of 343 turtles killed as they fi rst entered the highway adjacent to the shoulder and the remaining fi ve percent killed in the fi rst two traffi c lanes. I used the equation, Pkilled = 1 - e -Na/v, to estimate the probability of being struck in one attempted crossing of U.S. Highway 27, where N is traffi c rate in vehicles/lane/sec during 80 percent of daily volume, a = width of the kill zone (2 tire widths per lane plus 2 times weighted average shell length of 5 species), and v = turtle velocity (m/sec). Solving this equation results in a 98 percent probability of a turtle being killed in one attempted crossing, closely matching my direct observations. Using this model and historic traffi c data, the probability of a turtle successfully crossing U.S. Highway 27 decreased from 32 percent in 1977 to only two percent in 2001. Therefore, at least 98 percent of turtles diverted by the fences would have been killed on U.S. Highway 27 during this study if fences were not in place, and the road kill rate is estimated at 1,294/km/yr. Based on a literature survey, this is the highest attempted crossing rate ever documented for turtles. Sex ratios (M:F) of Pseudemys fl oridana (4:1), Trachemys scripta (3:1), and Sternotherus odoratus (2:1) were signifi cantly male-biased and low numbers of mature females are likely due to 5-10 percent annual road mortality during attempted nesting forays. Because of demographic and life history constraints, turtle populations cannot compensate for the combined effects of annual road mortality (5-10%) and periodic mass road mortality (95-99%) during lake dry-downs.