Nafenopin was administered orally for 21 days to male Sprague-Dawley rats (0.5-50 mg/kg/day), Syrian hamsters (5-250 mg/kg/day), Dunkin-Hartley guinea pigs (50 and 250 mg/kg/day), and marmosets (Callithrix jacchus, 50 and 250 mg/kg/day). With the rat, and to a lesser extent in the hamster, nafenopin treatment produced dose-related increases in liver size and induction of peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidizing enzyme activities. In contrast, in the guinea pig and marmoset, there was no effect on liver size and only comparatively small changes were observed in these enzyme activities. Ultrastructural examination of liver sections from nafenopin-treated rats and hamsters revealed increased numbers of peroxisomes many of which lacked the characteristic crystalline nucleoid. While nafenopin had little effect on peroxisome numbers in either the guinea pig or marmoset, increases in microsomal cytochrome P450 content and mixed function oxidase activities were observed in these species. These results demonstrate marked species differences in nafenopin-induced hepatic peroxisome proliferation with the Syrian hamster being less responsive than the rat and the guinea pig and marmoset being only weakly responsive. As nafenopin is a known hepatocarcinogen in the rat, comparative long-term studies in poorly responsive species, such as the guinea pig and marmoset, may help clarify the role of organelle proliferation in the hepatocarcinogenicity of certain peroxisome proliferators.