The plant growth regulator, abscisic acid (ABA), is synthesized via the oxidative cleavage of an epoxy-carotenoid. Specifically, a double bond is cleaved by molecular oxygen and an aldehyde is formed at the site of cleavage in both products. The Vp14 gene from maize encodes an oxidative cleavage enzyme for ABA biosynthesis and the recombinant VP14 protein catalyzes the cleavage reaction in vitro. The enzyme has a strict requirement for a 9-cis double bond adjacent to the site of cleavage (the 11-12 bond), but shows some plasticity in other features of carotenoids that are cleaved. A kinetic analysis with the 9-cis isomer of five carotenoids displays several substrate activity relationships. One of the carotenoids was not readily cleaved, but inhibited the cleavage of another substrate in mixed assays. Of the remaining four carotenoids used in this study, three of the substrates have similar V(max) values. The V(max) for the cleavage of one carotenoid substrate was significantly higher. Molecular modeling and several three-dimensional quantitative substrate-activity relationship programs were used to analyze these results. In addition to a 9-cis double bond, the presence and orientation of the ring hydroxyl affects substrate binding or the subsequent cleavage. Additional variations that affect substrate cleavage are proposed.