Lifestyle factors such as smoking have been shown to influence urinary mutagenicity. Therefore, these factors have to be considered carefully when evaluating occupational genotoxic exposures. We investigated day-to-day variability in active and passive tobacco smoke exposure by studying urinary cotinine levels and determined their influence on observed urinary mutagenicity. Urinary cotinine was assessed for 105 subjects employed in the rubber manufacturing industry in the Netherlands on Sunday, Wednesday, and Thursday. Urinary mutagenicity was measured by the Salmonella typhimurium strain YG1041 with metabolic activation for the Sunday urine sample and a pooled weekday urine sample. A sharp decrease in urinary cotinine concentration was observed during the week compared to Sunday for smokers (39%; P < 0.01) and nonsmokers (23%). Different smoking habits on Sunday resulted in higher regression coefficients for categorical proxies for smoking habits and urinary mutagenicity levels. However, regression coefficients for urinary cotinine and urinary mutagenicity were similar for the Sunday and weekday urine samples (beta = 0.29 and beta = 0.28, respectively). Consequently, these estimates were used to adjust urinary mutagenicity for tobacco smoke intake. Cotinine-adjusted urinary mutagenicity levels were comparable between smokers and nonsmokers, and a similar increase in urinary mutagenicity of 39% and 34%, respectively, was observed for both smokers and nonsmokers due to occupational genotoxic exposures or other changes in lifestyle factors. These results indicate that the introduction of nonsmoking policies in the workplace has reduced exposure to mainstream and environmental tobacco smoke, resulting in a temporal variation in lifestyle-related mutagenicity. Therefore, adequate adjustment for daily tobacco smoke exposure is a necessity when using the urinary mutagenicity assay to evaluate possible genotoxic exposures in the workplace.