The mutagenicity of urine obtained from five cigarette smokers was investigated using two bacterial assays: the Ames test and the SOS Chromotest. Urinary mutagens were extracted on Amberlite XAD-2 resin. Four urine samples showed activity towards Salmonella typhimurium tester strain TA98 with S9 mix while no SOS-inducing activity could be measured with Escherichia coli strain PQ37 in the SOS Chromotest. Using factorial design and a positive control benzo[a]pyrene (BaP), the concentration of S9, nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P) were optimized (2%, 0.5 mM and 10 mM respectively) for the SOS Chromotest. The SOS-inducing power of BaP was 1.42/nM with the standard S9 mix and 3.26/nM with the optimized S9 mix. B buffer and the age of L-broth were found to decrease the sensitivity of beta-galactosidase assays in the SOS Chromotest. A 4000-fold urine concentrate from a smoker was finally tested using the Ames test and the modified SOS Chromotest. Mutagenic and toxic activities were found toward tester strain TA98 (+S9 mix) showing that the SOS Chromotest is not at present suitable for assaying urinary mutagens in the presence of an in vitro metabolic activating mixture.