Cigarette smokers have been reported to void urine which is more mutagenic, as measured in the Ames assay, than urine voided by non-smokers. Condensate from the mainstream smoke of a cigarette which primarily heats tobacco (test cigarette) has shown significantly reduced mutagenicity in a battery of in vitro genotoxicity assays compared with tobacco-burning cigarettes. The objective of this study was to determine whether the reduction in mutagenic activity observed in the in vitro assays would be reflected in the urine of smokers of the test cigarette. Twenty smokers were enrolled in a 4-week crossover study, with each smoker consuming test cigarettes ad libitum for a week and their usual brand of tobacco-burning cigarettes the other 3 weeks. Diet was strictly controlled throughout the study, and broiled and pan-fried meat was not served to minimize ingestion of mutagenic protein pyrolysis products. There was no statistically significant difference (p = 0.06) in consumption of tobacco-heating and tobacco-burning cigarettes. There were no statistically significant differences (p = 0.22) in salivary cotinine concentrations for smokers when smoking either tobacco-burning or tobacco-heating cigarettes. Urinary nicotine (ng/mg creatinine) was not different (p = 0.31) for smokers when smoking either tobacco-burning or tobacco-heating cigarettes. Urinary cotinine (ng/mg creatinine) was 32% lower (p = 0.0004) when smoking tobacco-heating cigarettes as compared with smoking tobacco-burning cigarettes. Twenty-four-hour urine samples were collected twice weekly, concentrated using XAD-2 resin and tested in Ames strains TA98 and YG1024 with metabolic activation. Tobacco-burning cigarette smokers experienced a 79% reduction in urinary mutagenicity as measured in strain YG1024 and a 72% reduction as measured in strain TA98 during the week that they smoked the tobacco-heating cigarette while maintaining a fixed dietary regimen. The results of this study indicate that smokers of tobacco-heating cigarettes void urine which is significantly less mutagenic than urine voided by smokers of tobacco-burning cigarettes.