Acrylonitrile is a heavily produced unsaturated nitrile, which is used in the production of synthetic fibers, plastics, resins, and rubber. Acrylonitrile is a multisite carcinogen in rats after exposure via gavage, drinking water, or inhalation. No carcinogenicity studies of acrylonitrile in a second animal species were available. The current studies were designed to assess the carcinogenicity of acrylonitrile in B6C3F1 mice of both sexes. Acrylonitrile was administered by gavage at 0, 2.5, 10, or 20 mg/kg/day, 5 days per week, for 2 years. Urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine were measured as markers of exposure to acrylonitrile. In general, there were dose-related increases in urinary thiocyanate and N-acetyl-S-(2-cyanoethyl)-L-cysteine concentrations in all dosed groups of mice and at all time points. Survival was significantly (p < 0.001) reduced in the top dose (20 mg/kg) group of male and female mice relative to controls. The incidence of forestomach papillomas and carcinomas was increased in mice of both sexes in association with an increase in forestomach epithelial hyperplasia. The incidence of Harderian gland adenomas and carcinomas was also markedly increased in the acrylonitrile-dosed groups. In female mice, the incidence of benign or malignant granulosa cell tumors (combined) in the ovary in the 10 mg/kg dose group was greater than that in the vehicle control group, but because of a lack of dose response, this was considered an equivocal finding. In addition, the incidences of atrophy and cysts in the ovary of the 10 and 20 mg/kg dose groups were significantly increased. The incidences of alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in female mice treated with acrylonitrile at 10 mg/kg/day for 2 years. This was also considered an equivocal result. In conclusion, these studies demonstrated that acrylonitrile causes multiple carcinogenic effects after gavage administration to male and female B6C3F1 mice for 2 years.