The induction of liver fatty acid binding protein (L-FABP) by the peroxisome proliferators bezafibrate and clofibrate was compared with the induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation in cultured rat hepatocytes maintained on a substratum of laminin-rich (EHS) gel. This substratum was chosen because marked induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was effected by bezafibrate in hepatocytes supported on EHS gel, whereas only peroxisomal palmitoyl-CoA oxidation was induced in hepatocytes maintained on collagen-coated plates. In control cells on EHS, activity of peroxisomal palmitoyl-CoA oxidation remained stable, while L-FABP abundance declined with time, and L-FABP mRNA was undetectable after 5 days. In cultures exposed to bezafibrate or clofibrate, peroxisomal palmitoyl-CoA oxidation activity was induced earlier and more rapidly than L-FABP. When fibrates were withdrawn, peroxisomal palmitoyl-CoA oxidation declined rapidly, whereas L-FABP continued to increase. L-FABP induction was accompanied by a striking increase in mRNA specifying this protein. Tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I, effectively doubled peroxisomal palmitoyl-CoA oxidation activity. However, tetradecylglycidic acid markedly inhibited fibrate induction of L-FABP and peroxisomal palmitoyl-CoA oxidation but, unexpectedly, did not prevent the fibrate-induced proliferation of peroxisomes. Maximal induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was produced at a bezafibrate concentration in the culture medium (0.05 mM) much lower than that of clofibrate (0.3 mM). Also, bezafibrate, but not clofibrate, inhibited [1-14C]oleic acid binding to L-FABP with a Ki = 9.5 microM. We conclude that hepatocytes maintained on EHS gel provide an important tool for investigating the regulation of L-FABP. These studies show that the induction of peroxisomal beta-oxidation and L-FABP by peroxisome proliferators are temporally consecutive but closely related processes which may be dependent on a mechanism distinct from that which leads to peroxisome proliferation. Furthermore, the mechanism of action of the more potent peroxisome proliferator, bezafibrate, may be mediated, in part, by interaction of this agent with L-FABP.