Fumonisin B 1 (FB 1), a mycotoxin produced by a common corn contaminant Fusarium moniliforme and a hepatocarcinogen in rats, has been previously suggested to act as a poor initiator, but a better promoter of γ-glutamyltranspeptidase (GGT)-positive rat liver preneoplastic lesions. Using glutathione S-transferase-placental form (GSTP) as a more sensitive marker of initiation, we have further evaluated the initiating capacity of various doses of purified FB 1 administered (a) intraperitoneally (IP) to male Sprague–Dawley (SD) rats for 4 days and (b) orally (PO) to male and female SD rats for 11 days. Compared to their respective controls, significant increases in GSTP-positive hepatocytes were observed in male rats administered FB 1 IP at 10 mg/kg body weight/day for 4 days, as well as in male and female rats treated with 35 and 75 mg/kg body weight/day FB 1 PO for 11 days. The percentage section area of liver occupied by GSTP-positive mini-foci comprising of three to 12 cells was increased significantly in male rats given 10 mg/kg FB 1 IP, or in PO-treated males and females with 75 mg/kg FB 1. Both IP and PO FB 1 treatments resulted in dose-related enhanced hepatocyte proliferation as measured by proliferating cell nuclear antigen (PCNA) labeling with significant increases in the number of PCNA-positive nuclei at the same IP and PO dose levels where the number of GSTP-positive cells were elevated. In all studies, enhanced PCNA and GSTP expression occurred at FB 1 doses which, based on serum biochemical and histopathological data previously reported from our laboratory, were shown to be hepatotoxic. Therefore, our data suggest that in a manner similar to known genotoxic carcinogens, FB 1 has the capacity to initiate GSTP-positive hepatocytes with their subsequent development into GSTP mini-foci at exposure levels that induce enhanced hepatocyte proliferation in response to liver toxicity. In SD rats, this occurs as early as within 4 days of IP treatment or 11 days of PO treatment.