3,30,4,40-tetrachloroazobenzene (TCAB) is a contaminant formed during manufacture of various herbicide compounds. A recent National Toxicology Program study showed B6C3F1 mice exposed to TCAB developed a treatment-related increase in lung carcinomas in the high-dose group, and urethral carcinomas, an extremely rare lesion in rodents, in all dose groups. As the potential for environmental exposure to TCAB is widespread, and the mechanisms of urethral carcinogenesis are unknown, TCAB-induced urethral and pulmonary tumors were evaluated for alterations in critical human cancer genes, Kras and Tp53. Uroplakin III, CK20, and CK7 immunohistochemistry was performed to confirm the urothelial origin of urethral tumors. TCAB-induced urethral carcinomas harbored transforming point mutations in K-ras (38%) and Tp53 (63%), and 71%displayed nuclear TP53 expression, consistent with formation of mutant protein. Transition mutations accounted for 88%of Tp53 mutations in urethral carcinomas, suggesting that TCAB or its metabolites target guanine or cytosine bases and that these mutations are involved in urethral carcinogenesis. Pulmonary carcinomas in TCAB-exposed animals harbored similar rates of Tp53 (55%) and Kras (36%) mutations as urethral carcinomas, suggesting that TCAB may induce mutations at multiple sites by a common mechanism. In conclusion, TCAB is carcinogenic at multiple sites in male and female B6C3F1 mice through mechanisms involving Tp53 and Kras mutation.