In plant glyoxysomes, an enzyme activity responsible for a particular step in the fatty acid beta-oxidation is located on more than one protein species. Various monofunctional enzymes and two forms of a multifunctional protein are involved in the degradation of cis-unsaturated fatty acids. delta 3, delta 2-Enoyl-CoA isomerase activity, previously found to be located on a monofunctional dimeric protein, is attributable to one form of the monomeric multifunctional protein (MFP). The presence or absence of isomerase activity allows us to differentiate between the tetrafunctional 76.5-kDa isoform (MFP II) and the trifunctional 74-kDa isoform (MFP I) in cucumber (Cucumis sativus) cotyledons. Both MFP I and MFP II exhibited blocked N-terminal structures. MFP I and MFP II are distinguishable from each other by their susceptibility to limited proteolysis. A series of examples is presented describing the preparation of enzymically active proteolytic fragments. We demonstrate that both forms of the monomeric MFP are composed of domains separable from each other without loss of activity. By fragmentation of MFP I and subsequent chromatography, a 60-kDa peptide was purified retaining hydratase and epimerase activity but lacking dehydrogenase activity. In addition, a highly positively charged fragment was observed carrying solely dehydrogenase activity. From MFP II, a 36-kDa fragment with hydratase activity was characterized. An enzymically inactive 46-kDa fragment was prepared from MFP II and sequenced at its unblocked N-terminus.