Riddelliine (RID) is a pyrrolizidine alkaloid found in plants of the genera Crotalaria, Amsinckia, and Senecio in the United States. RID has been extensively studied in a wide variety of in vitro short-term genotoxicity tests and has yielded positive responses in most test systems; however, there are fewer data available on the effects of RID in in vivo assays and no data under repeat-dose regimens. We have evaluated the ability of RID to induce unscheduled DNA synthesis (UDS) in hepatocytes, S-phase synthesis (SPS) in hepatocytes, and micronuclei in bone marrow from animals dosed for 5 or 30 days in conjunction with prechronic toxicity testing conducted for the National Toxicology Program. Results of this study indicate that RID did not induce an increase in micronucleated polychromatic erythrocytes (PCE) in bone marrow of mice after 5 days of dosing or in PCE from rats or mice after 30 days of dosing. RID did not induce an increase in UDS in rat hepatocytes after 5 or 30 days of dosing, but it did induce an equivocal UDS response in male mice after both time points and a positive response in female mice after 30 days of dosing. RID induced significant elevations in SPS in rat hepatocytes after both 5 and 30 days of dosing, even at low doses. An increase in SPS was observed in male and female mouse hepatocytes, but only after 30 days of dosing. Rats and mice of both sexes showed a depression in SPS at higher doses. This effect may be a result of toxicity, which compromises the ability of the liver to regenerate. These results demonstrate that repeated administration of test chemicals may alter the genotoxic response to chemicals.