The aim of this work was to investigate the stereoselectivity of maize leaf phosphoenolpyruvate carboxylase with E- and Z-2-phosphoenolbutyrate as inhibitors and substrates. In addition, a procedure is presented for the separation of the isomers of 2-phosphoenolbutyrate. The method is based on the different interaction of those compounds with a strong anion-exchange high-pressure liquid chromatography column using 50 mM potassium phosphate (pH 3) as elution buffer, and allows the obtention of pure E- and Z-P-enolbutyrate with high yield. The same system was used to identify Z-P-enolbutyrate as the product of the phosphorylation of 2-oxobutyrate by rabbit muscle pyruvate kinase. In the presence of 5 mM Mg2+, both isomers of P-enolbutyrate inhibited C4-plant P-enolpyruvate carboxylase; the values of Ki were 15-20 microM and 100-110 microM for Z- and E-P-enolbutyrate, respectively. With 0.5 mM Mn2+, the Z isomer was also effective as inhibitor (Ki = 35-40 microM), while the E isomer produced activation of the carboxylase probably due to its binding at an allosteric site. Both compounds were substrates of the enzyme with similar V/Km values; however, V and Km for the two isomers were significantly different (i.e. Km = 110 microM for Z-P-enolbutyrate and 220 microM for E-P-enolbutyrate). The results indicate the existence of stereoselectivity for the binding of P-enolbutyrate to the active site of P-enolpyruvate carboxylase. However, this fact does not affect the use of the isomers as substrates by the plant carboxylase.