Menthol is widely used in the pharmaceutical, cosmetic, food and tobacco industries and is generally regarded as sale (GRAS) for these applications. Menthol itself is non-mutagenic in the Ames Assay. This study was conducted to compare the mutagenicity of wet total particulate matter (WTPM) and gas-vapor phase (GVP) as well as whole-smoke from a non-mentholated and mentholated cigarette to determine if the presence of menthol has an effect on this biological endpoint. The cigarettes used were comparable in construction, composition, and WTPM deliveries. The mentholated cigarette contained menthol at a normal user level. Cigarettes were smoked with a 35mL puff volume, 2 second puff duration and a 1 minute puff interval on a VITROCELL→ VC10 smoking robot. WTPM from each cigarette type was pad collected and extracted in dimethylsulfoxide. Salmonella strains TA98 and TA100 were exposed to WTPM with metabolic activation (S9+). Whole-smoke (TA98 and TA100, S9+) and GVP (TA100, S9-) exposures utilized the VITROCELL→ exposure modules and smoke dilution system. No differences were detected in the specific activities (revertants / µg) of the WTPM (TA98, p = 0.8335; TA100, p = 0.7889) or the GVP (p = 0.4595). Differences were observed in the whole-smoke activities, with the mentholated sample having significantly lower specific activity, less mutagenic, than the non-mentholated cigarette (TA98, p = 0.0027; TA100, p = 0.0297), only from cigarettes used from a freshly opened pack. No differences in whole-smoke cytotoxicity were observed. No differences in whole-smoke activities occurred when cigarettes were opened and conditioned (23°C, 60% RH) for approximately 18 hours prior to smoking. Analytical data suggests these results may be partially explained by the loss of menthol upon cigarette conditioning.