In human tissues phytanic acid is alpha-oxidized to pristanic acid in peroxisomes. Studies of the intraorganellar site of alpha-oxidation of [1-14C]phytanic acid to pristanic acid in peroxisomes isolated from human liver demonstrate that phytanoyl-CoA ligase is present in the peroxisomal membrane and that the enzyme system for alpha-oxidation of phytanic acid to pristanic acid is in the peroxisomal matrix. In contrast to the beta-oxidation system for fatty acids, the substrate for alpha-oxidation is free phytanic acid. The studies described in this manuscript report a novel fatty acid oxidation system where the substrate for the enzyme system is free fatty acid; however, phytanoyl-CoA ligase regulates the alpha-oxidation of phytanic acid at the organellar (peroxisomal) level.