Acyl-CoA binding protein (ACBP) and fatty acid binding protein (FABP) are important intracellular lipid binding proteins. The purpose of the present experiments was to test the hypothesis that peroxisome proliferators induce ACBP in rat hepatocytes as has been shown previously for FABP. The effects of two structurally dissimilar peroxisome proliferators perfluorodecanoic acid (PFDA) and clofibric acid (CPIB) were examined in primary rat hepatocyte cultures in a chemically defined media. Both compounds alter lipid metabolism in primary rat hepatocytes in a similar fashion, although PFDA is more potent than CPIB at inducing peroxisomal beta-oxidation. In addition, PFDA and CPIB compete with long-chain fatty acids for binding to FABP but do not compete with long-chain acyl-CoA esters for binding to ACBP. The concentration of ACBP and FABP was increased in peroxisome proliferator-treated hepatocytes relative to vehicle controls within 48 h of treatment. Evidence is given to support increases in ACBP and FABP mRNA being the cause of the increased protein levels by peroxisome proliferators. In addition, the peroxisome proliferators PFDA, perfluorooctanoic acid and ciprofibrate induced hepatic ACBP following in vivo administration to rats indicating that this phenomena is not exclusive to in vitro systems. Therefore, ACBP appears to be a member of the peroxisome proliferator loci, a group of lipid metabolizing proteins, including FABP, which are regulated by peroxisome proliferators such as fibric acids and perfluorinated fatty acids.