Acrylonitrile was tested for reproductive effects in a three generation drinking water study with two matings per generation. Sprague–Dawley rats were exposed to acrylonitrile in drinking water at 0, 100, or 500 ppm. This corresponds to 0, 11±5 and 37±10 mg/kg, respectively, for males and 0, 20±3 and 40±8 mg/kg per day for the females, respectively. Water consumption was reduced in F0 rats in the 100 and 500 ppm groups. At 500 ppm, acrylonitrile reduced body weight gain and food intake of the first generation parental rats (F0). These parameters were not investigated at subsequent generations. The pup survival (both viability and lactation indices) was reduced at the 500 ppm treatment level in both matings of all three generations. Fostering the 500 ppm pups onto untreated mothers following the second mating lessened mortality, suggesting a maternal effect consistent with decreased water consumption. There was no remarkable change in the reproductive capacity in any of matings in rats at the 100 ppm concentration. In contrast, in all three generations, the body weights of the pups of the 500 ppm treatment level were reduced on Day 21 at both matings. No adverse findings were observed in the tissues of a limited number of third generation weanlings (F3b) upon gross and microscopic evaluation. No effect on the sciatic nerve was evident among the adult female rats held for 20 weeks after weaning of the second litter. There was a dose-related effect of acrylonitrile on gross masses in female rats at each parental generation held 20 weeks after the weaning of the second litter. Histopathological evaluation of these dams showed an increase in astrocytomas and zymbal gland tumors.