In Refsum disease, disorders of peroxisome biogenesis, and rhizomelic chondrodysplasia punctata, pathological accumulation of phytanic acid results from impaired alpha-oxidation of this branched-chain fatty acid. Previous studies from this laboratory indicated that activation of phytanic acid to its CoA derivative precedes its alpha-oxidation in peroxisomes. It was reported that this reaction is catalyzed by a unique phytanoyl-CoA synthetase in human peroxisomes. We wanted to determine whether phytanic acid activation in rats required long-chain acyl-CoA synthetase (LCS), very long-chain acyl-CoA synthetase (VLCS), or a different enzyme. To test directly whether LCS could activate phytanic acid, rat liver cDNA encoding this enzyme was transcribed and translated in vitro. The expressed enzyme had both LCS activity (assayed with palmitic acid, C16: 0) and phytanoyl-CoA synthetase activity; VLCS activity (assayed with lignoceric acid, C24: 0) was not detectable. The ratio of phytanoyl-CoA synthetized activity to palmitoyl-CoA synthetase activity for LCS synthetized in vitro (approximately 205) was higher than that observed in peroxisomes isolated from rat liver (5-10%), suggesting that the expressed enzyme contained sufficient phytanoyl-Coa synthetase activity to account for all activity observed in intact peroxisomes. Further experiments were carried out to verify that phytanic acid was activated by LCS in rat liver peroxisomes. Attempts to separate LCS from phytanoyl-CoA synthetase by chromatography on several matrices were unsuccessful. Preparative isoelectric focusing revealed that phytanoyl-CoA synthetase and LCS had indistinguishable isoelectric points. Phytanoyl-CoA synthetase activity was inhibited by unlabeled palmitic acid but not by lignoceric acid. Heat treatment inactivated both phytanoyl-CoA and palmitoyl-CoA synthetase activities at similar rates. 5,8,11,14-Eicosatetraynoic acid inhibited activation of phytanic acid and palmitic acid in a parallel dose-dependent manner, whereas activation of lignoceric acid was not affected. These data support our conclusion that rat liver LCS, an enzyme known to be present in peroxisomal membranes, has phytanoyl-CoA synthetase activity.