Peroxisomes have been shown to play an important role in the oxidative degradation of (poly)unsaturated fatty acids, and contain the enzyme activities needed for the metabolism of double bonds of unsaturated fatty acids in connection with this physiological function. Our understanding of the metabolic pathways and enzyme activities involved in the degradation of unsaturated acyl-CoAs has undergone a re-evaluation recently, and though many open questions still remain significant progress has been made, especially concerning the reactions metabolizing double bonds. The enzyme activities to be discussed here are 2,4-dienoyl-CoA reductase; 3/2-enoyl-CoA isomerase; 2-enoyl-CoA hydratase 2; 5-enoyl-CoA reductase and 3,5/2,4-dienoyl-CoA isomerase. Some of these activities are integral parts of the multifunctional proteins of beta-oxidation systems, which must also be taken into account in this context.