It is unknown whether peroxisome proliferators decrease hepatic fatty acid oxidation via uncoupling of respiration or if they inhibit extramitochondrial fatty acyl CoA synthesis. Therefore, the purpose of this study was to examine both processes simultaneously using the isolated perfused liver, a whole cell preparation where enzymes and biochemical processes can be monitored continuously under nearly physiological conditions. Accordingly, ketone body formation (beta-hydroxybutyrate + acetoacetate) from lipid metabolism and oxygen uptake, which is increased by uncoupling agents, were monitored at the same time. 2-Bromooctanoate, a known inhibitor of acyl CoA synthetase, decreased ketone body formation in a dose-dependent manner without altering cellular respiration (half-maximal inhibition, approximately 25 microM) and concomitantly increased protein kinase C nearly fourfold also in a dose-dependent fashion. Ketogenesis was also blocked maximally 50-66% with mono(ethylhexyl)phthalate, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (WY-14,643), and nafenopin, potent peroxisome proliferators and tumor promoters. These compounds also increased protein kinase C three- to fourfold without altering oxygen uptake significantly. Thus, lipid metabolism appears to be the prime target of potent peroxisome proliferators most likely on actions via acyl CoA synthetase rather than oxidative phosphorylation. In contrast, weak peroxisome proliferators and tumor promoters, di(ethylhexyl)phthalate and 2-ethylhexanol, did not affect ketogenesis, oxygen consumption, or protein kinase C at similar concentrations. Additionally, octanoate increased ketone body formation in the presence of nafenopin. Because octanoate is metabolized by mitochondrial acyl CoA synthetase independent of carnitine acyltransferase, these results indicate that nafenopin does not inhibit mitochondrial beta-oxidation. Taken together, it is concluded that potent peroxisome proliferators preferentially block ketogenesis without altering cellular respiration in the liver. This phenomenona, which occurs due to inhibition of acyl CoA synthetase, leads to an elevation of free fatty acids that stimulates protein kinase C and promotes cell proliferation.