The aim of the present study was to investigate the hepatic regulation and beta-oxidation of long-chain fatty acids in peroxisomes and mitochondria, after 3-thia- tetradecylthioacetic acid (C14-S-acetic acid) treatment. When palmitoyl-CoA and palmitoyl-L-carnitine were used as substrates, hepatic formation of acid-soluble products was significantly increased in C14-S-acetic acid treated rats. Administration of C14-S-acetic acid resulted in increased enzyme activity and mRNA levels of hepatic mitochondrial carnitine palmitoyltransferase (CPT)-II. CPT-II activity correlated with both palmitoyl-CoA and palmitoyl-L-carnitine oxidation in rats treated with different chain-length 3-thia fatty acids. CPT-I activity and mRNA levels were, however, marginally affected. The hepatic CPT-II activity was mainly localized in the mitochondrial fraction, whereas the CPT-I activity was enriched in the mitochondrial, peroxisomal, and microsomal fractions. In C14-S-acetic acid-treated rats, the specific activity of peroxisomal and microsomal CPT-I increased, whereas the mitochondrial activity tended to decrease. C14-S-Acetyl-CoA inhibited CPT-I activity in vitro. The sensitivity of CPT-I to malonyl-CoA was unchanged, and the hepatic malonyl-CoA concentration increased after C14-S-acetic acid treatment. The mRNA levels of acetyl-CoA carboxylase increased. In hepatocytes cultured from palmitic acid- and C14-S-acetic acid-treated rats, the CPT-I inhibitor etomoxir inhibited the formation of acid-soluble products 91 and 21%, respectively. In contrast to 3-thia fatty acid treatment, eicosapentaenoic acid treatment and starvation increased the mitochondrial CPT-I activity and reduced its malonyl-CoA sensitivity. Palmitoyl-L-carnitine oxidation and CPT-II activity were, however, unchanged after either EPA treatment or starvation. The results from this study open the possibility that the rate control of mitochondrial beta-oxidation under mitochondrion and peroxisome proliferation is distributed between an enzyme or enzymes of the pathway beyond the CPT-I site after 3-thia fatty acid treatment. It is suggested that fatty acids are partly oxidized in the peroxisomes before entering the mitochondria as acylcarnitines for further oxidation.