Primary cultures of rat hepatocytes were exposed to a range of chemicals known to cause peroxisome proliferation in vivo. Peroxisomal palmitoyl-CoA oxidation and carnitine acetyltransferase (CAT) activity were increased within 12 to 24 hr of adding 0.5 mM clofibrate to the culture medium, reaching about 20 times control levels after 72 hr. Stimulation of CAT activity was dose related over a concentration range of 0.05 to 2 mM clofibrate and 0.02 to 0.2 mM mono-2-ethylhexylphthalate (MEHP). Higher concentrations of MEHP were cytotoxic. The stimulation of CAT activity and palmitoyl-CoA oxidation produced by clofibrate and MEHP was inhibited by cycloheximide. In further studies with clofibrate and a range of other known peroxisome proliferators (nafenopin, tiadenol, BR-931, Wy-14,643, and acetylsalicylic acid), induction of CAT and palmitoyl-CoA oxidation was observed with no increase in activity of another peroxisomal enzyme, D-amino acid oxidase. This differential effect on peroxisomal enzyme activity is typical of that seen in vivo. Furthermore, the relative potencies of the different peroxisome proliferators in vitro agreed well with what is known from studies in vivo. Mitochondrial and microsomal marker enzymes showed little change in activity. Electron microscopy of treated cultures revealed increased numbers of peroxisomes, some of which lacked the characteristic nucleoid. The results indicate that primary cultures of rat hepatocytes provide a rapid, sensitive means of identifying chemicals that cause peroxisome proliferation and a potentially valuable system for studies aimed at clarifying the toxicological significance of this phenomenon.