The alpha-oxidation of phytanic acid in rat liver is a mitochondrial function. The inhibition of phytanic acid oxidation activity by inhibitors of acyl-CoA ligases (Naproxen and Triacsin C) and that of carnitine acyltransferase I (2-(5-(4-chlorophenyl)pentyl)oxirane-2 carboxylic acid (POCA) and 2-bromopalmitate) and increase in phytanic acid oxidation activity by the addition of exogenous carnitine and CoA to purified mitochondria suggests that phytanoyl-CoA ligase and carnitine acyltransferase I are essential for the activation and transport of phytanic acid across the mitochondrial membrane. This was further supported by the fact that activation of phytanic acid to phytanoyl-CoA was required only in intact mitochondria but not in mitochondria permealized with digitonin. DesulfoCoA, Naproxen and POCA treatment resulted in a significant decrease in phytanic acid oxidation in intact mitochondria but not in digitonin permealized mitochondria. These results show that alpha-oxidation of phytanic acid to pristanic acid, in contrast to beta-oxidation of fatty acids, requires free fatty acid as substrate. The inhibition of alpha-oxidation (approximately 90%) of phytanic acid by different cytochrome P-450 enzyme inhibitors indicated that alpha-oxidation of phytanic acid is mediated through cytochrome P-450 containing enzyme system. Similar to the omega-hydroxylation system in endoplasmic reticulum, alpha-hydroxylation and the subsequent alpha-oxidation of phytanic acid in mitochondria is induced by ciprofibrate, a hypolipidemic drug.