To understand the possible role of phytanoyl-CoA ligase, present in the membrane, in the oxidation of phytanic acid in the matrix of peroxisomes (Pahan, K. and I. Singh. 1993. FEBS Lett. 333: 154-158) we examined the transport of phytanic acid/phytanoyl-CoA into peroxisomes and the topology of the active site of phytanoyl-CoA ligase in the peroxisomal membrane. The increase in lignoceroyl-CoA ligase as compared to no change in the activities of palmitoyl-CoA and phytanoyl-CoA ligases when peroxisomes were disrupted with detergent or sonication and inhibition of the activities of both palmitoyl-CoA and phytanoyl-CoA ligase by impermeable inhibitor of acyl-CoA ligases (mercury-dextran) and trypsin treatment in the intact peroxisomes. On the other hand, the lignoceroyl-CoA ligase activity was inhibited by mercury-dextran and trypsin only in the disrupted peroxisomes. Taken together, these studies support the conclusion that the enzymatic site of phytanoyl-CoA ligase is on the cytoplasmic surface of peroxisomal membrane. This implies that phytanoyl-CoA is synthesized on the cytoplasmic surface of peroxisomal membrane and is translocated through the membrane for its alpha-oxidation to pristanic acid in the matrix of peroxisomes. To delineate the transport for phytanic acid through the peroxisomal membrane, we examined cofactors and energy requirements for its transport into peroxisomes. The similar rates of transport of phytanoyl-CoA and phytanic acid under conditions favorable for fatty acid activation (presence of ATP, CoASH, and MgCl2) and the lack of transport of phytanic acid when ATP and/or CoASH were removed or replaced with their inactive analogues (ATP and/or CoASH) from assay medium clearly demonstrates that the transport of phytanic acid requires prior synthesis of phytanoyl-CoA by phytanoyl-CoA ligase. The prerequisite activation of phytanic acid to phytanoyl-CoA for its alpha-oxidation only in intact peroxisomes, and oxidation of free phytanic acid in digitonin-permealized peroxisomes or isolated matrix, suggests that phytanoyl-CoA ligase (in peroxisomal membrane) regulates the oxidation of phytanic acid in peroxisomes by providing phytanoyl-CoA for its transport into peroxisomes.