Recently, it has become clear that the peroxisomal beta-oxidation system in rat and man consists of multiple pathways. In rat and man straight chain fatty acids, dicarboxylic fatty acids and prostaglandins are oxidized via the L-specific pathway catalyzed by palmitoyl-CoA oxidase, multifunctional protein-1 and thiolase. 2-Methyl-branched fatty acids and the bile acid intermediates are oxidized via the D-specific pathway. In the rat this pathway is catalyzed by prostanoyl-CoA oxidase, multifunctional protein-2 and sterol carrier protein-X (branched fatty acids) and by trihydroxycoprostanoyl-CoA oxidase, multifunctional protein-2 and sterol carrier protein-X (bile acid intermediates). In the human, branched fatty acids and bile acid intermediates are oxidized via branched chain acyl-CoA oxidase, multifunctional protein-2 and sterol carrier protein-X. All enzymes of these pathways have been purified, cloned and characterized. Also the reactions that constitute the alpha-oxidation pathway for 3-methyl-branched fatty acids, have recently been identified in rat and man. The revised pathway consists of the following reactions: 1) an activation reaction catalyzed by an acyl-CoA synthetase, that forms a 3-methylacyl-CoA; 2) a hydroxylation (dioxygenase) reaction catalyzed by a 3-methylacyl-CoA 2-hydroxylase, that converts the CoA ester to a 2-hydroxy-3-methylacyl-CoA; 3) a cleavage reaction catalyzed by a 2-hydroxy-3-methylacyl-CoA lyase, that releases a 2-methyl fatty aldehyde and formyl-CoA. The branched aldehyde is dehydrogenated by an aldehyde dehydrogenase to a 2-methyl branched fatty acid that can be degraded by peroxisomal beta-oxidation. Formyl-CoA is enzymatically hydrolyzed to formate which is then converted to CO2.