IMPORTANCE:The US Food and Drug Administration (FDA) has announced its intention to reduce the nicotine content in combustible cigarettes but must base regulation on public health benefits. Fast nicotine metabolizers may be at risk for increased smoking following a national nicotine reduction policy. We hypothesized that using reduced nicotine content (RNC) cigarettes would be associated with increases in smoking behaviors and exposure among smokers with a fast-but not slow-nicotine-metabolite ratio (NMR). OBJECTIVES:To examine the association of RNC cigarettes with smoking behaviors and biomarkers of exposure and to compare these associations in fast and slow metabolizers of nicotine based on the NMR. DESIGN SETTING AND PARTICIPANTS:A 35-day, 3-period, within-participant nonrandomized clinical trial was conducted at an academic medical center in Philadelphia, Pennsylvania. A 5-day baseline period using the smokers' preferred brand of cigarettes was followed by 2 consecutive 15-day periods using free investigational RNC cigarettes. A total of 100 daily, non-treatment-seeking, nonmenthol cigarette smokers (59 fast, 41 slow metabolizers) were recruited from December 24, 2013, to December 2, 2015. Data analysis was performed from December 12, 2016, to January 3, 2018. INTERVENTIONS:Two 15-day periods using cigarettes containing 5.2 mg (RNC1) and 1.3 mg (RNC2) of nicotine per gram of tobacco. MAIN OUTCOMES AND MEASURES:Smoking behaviors (number of cigarettes per day [CPD], total puff volume) and biomarkers of exposure (carbon monoxide [CO], urine total nicotine equivalents [TNE], and 4-[methylnitrosamino]-1-[3-pyridyl]-1-butanol [NNAL]). RESULTS:Smokers (73 [73.0%] men; 74 [74.0%] white; mean [SD] age, 43.02 [12.13] years; mean [SD] CPD, 17.31 [5.72]) consumed 2.62 (95% CI, 1.54-3.70) more CPD during the RNC1 period vs their preferred brand during baseline (P < .001) and approximated baseline CPD during the RNC2 period (mean difference, 0.96 [95% CI, -0.36 to 2.28]; P = .24). Additional outcome measures were lower during both RNC periods vs baseline (total puff volume, mean [95% CI]: RNC1, 537 mL [95% CI, 479-595 mL]; RNC2, 598 mL [95% CI, 547-649 mL] vs baseline, 744 mL [95% CI, 681-806 mL]; TNE, mean [95% CI]: RNC1, 30.9 nmoL/mg creatinine [95% CI, 26.0-36.6 nmoL/mg]; RNC2, 22.8 nmoL/mg creatinine [95% CI, 17.8-29.0 nmoL/mg] vs baseline, 54.6 nmoL/mg creatinine [95% CI, 48.1-62.1 nmoL/mg]; and NNAL, mean [95% CI]: RNC1, 229 pg/mg creatinine [95% CI, 189-277 pg/mg]; RNC2, 190 pg/mg creatinine [95% CI, 157-231 pg/mg] vs baseline, 280 pg/mg creatinine [95% CI, 231-339 pg/mg]; all P < .001). Carbon monoxide measures were similar across study periods (CO boost [SD], RNC1, 4.6 ppm [4.1-5.1 ppm]; RNC2, 4.2 ppm [3.7-4.6 ppm]; and baseline, 4.4 ppm [3.8-4.9 ppm]). The RNC cigarette associations did not differ by NMR. CONCLUSIONS AND RELEVANCE:Both RNC cigarettes were associated with decreased puffing and urinary biomarker exposure but not with decreased daily cigarette consumption or CO levels. The NMR did not moderate associations at the nicotine levels tested, suggesting that fast metabolizers may not be at greater risk of increasing use or exposure from these products should the FDA mandate an RNC standard for cigarettes.