The biosynthesis of 4,7,10,13,16-22:5 and 4,7,10,13,16,19-22:6 requires that when 6,9,12,15,18-24:5 and 6,9,12,15,18,21-24:6 are produced in microsomes they must move to peroxisomes for partial beta-oxidation. When the 24-carbon acids were incubated with peroxisomes, 22-carbon acids with their first double bond at position 4 accumulated as did those with their first two double bonds at the 2-trans-4-cis-positions (D. L. Luthria, S. B. Mohammed, and H. Sprecher, J. Biol. Chem. 271, 16020-16025, 1996; and B. S. Mohammed, D. L. Luthria, S. P. Baykousheva, and H. Sprecher, Biochem. J., 326, 425-430, 1997). In the study reported here we analyzed the acyl-CoAs that accumulated when peroxisomes were incubated with 5,8,11,14-20:4 and 6,9,12-18:3, a metabolite that would be produced via one cycle of arachidonate degradation via the pathway requiring both NADPH-dependent 2,4-dienoyl-CoA reductase and delta 3,5, delta 2,4-dienoyl-CoA isomerase. With both substrates the acyl-CoAs of 2-trans-4-10:2, 4-10:1, 2-trans-4,7,10-16:4, and 4,7,10-16:3 accumulated. These results further establish that the reductase catalyzes a control step in the peroxisomal degradation of unsaturated fatty acids. It was not possible to detect any 18- or 12-carbon acyl-CoA when arachidonate was the substrate, nor did any 12-carbon catabolite accumulate from 6,9,12-18:3. The fractional amount of 5,8-14:2 and arachidonate catabolized via the pathway using only the enzymes of saturated fatty acid degradation versus the pathway that also uses the reductase and the isomerase could thus not be estimated.