A delta 3, delta 2-enoyl-CoA isomerase was extracted from fat-degrading cotyledons of cucumber seedlings. The enzyme is required for the degradation of cis-unsaturated fatty acids, e.g. linoleic acid being present in the storage tissue of cucumber seedlings in high amounts. The delta 3, delta 2-enoyl-CoA isomerase was exclusively localized within peroxisomes. Its purification included chromatography on a hydrophobic matrix, a cation exchanger, and on hydroxylapatite. 17,000-fold purification yielded a protein of apparent homogeneity. The isomerase is a homodimer with a Mr of 50,000 and an isoelectric point of pH approximately 8.1. Delta 3, delta 2-Enoyl-CoA isomerase reversibly catalyzes the conversion of both cis-3-enoyl-CoA and trans-3-enoyl-CoA into trans-2-enoyl-CoA. The isomerase exhibited optimal activity at pH 9 and was active with 3-enoyl-CoA species having chain lengths of C6-C12, with cis-hexanoyl-CoA being the most effective substrate. The purified enzyme did not show enoyl-CoA hydratase activity.