Microbial communities in soil A horizons derive their carbon from several potential sources: organic carbon (C) transported down from overlying litter and organic horizons, root-derived C, or soil organic matter. We took advantage of a multi-year experiment that manipulated the 14C isotope signature of surface leaf litter inputs in a temperate forest at the Oak Ridge Reservation, Tennessee, USA, to quantify the contribution of recent leaf litter C to microbial respiration and biomarkers in the underlying mineral soil. We observed no measurable difference (<∼40‰ given our current analytical methods) in the radiocarbon signatures of microbial phospholipid fatty acids (PLFA) isolated from the top 10cm of mineral soil in plots that experienced 3 years of litterfall that differed in each year by ∼750‰ between high-14C and low-14C treatments. Assuming any difference in 14C between the high- and low-14C plots would reflect C derived from these manipulated litter additions, we estimate that <∼6% of the microbial C after 4 years was derived from the added 1-4-year-old surface litter. Large contributions of C from litter<1 year (or >4 years) old (which fell after (or prior to) the manipulation and therefore did not differ between plots) are not supported because the 14C signatures of the PLFA compounds (averaging 200-220‰) is much higher that of the 2004-5 leaf litter (115‰) or pre-2000 litter. A mesocosm experiment further demonstrated that C leached from 14C-enriched surface litter or the O horizon was not a detectable C source in underlying mineral soil microbes during the first eight months after litter addition. Instead a decline in the 14C of PLFA over the mesocosm experiment likely reflected the loss of a pre-existing substrate not associated with added leaf litter. Measured PLFA Δ14C signatures were higher than those measured in bulk mineral soil organic matter in our experiments, but fell within the range of 14C values measured in mineral soil roots. Together, our experiments suggest that root-derived C is the major (>60%) source of C for microbes in these temperate deciduous forest soils. © 2010 Elsevier Ltd.