Five multiple forms (forms 1-5) of mandelonitrile lyase (EC 4.1.2.10) which catalyze the decomposition of mandelonitrile to benzaldehyde and hydrogen cyanide have been extensively purified from seeds of black cherry (Prunus serotina Ehrh.) by concanavalin A-Sepharose 4B chromatography and chromatofocusing. These forms are monomers which differ only slightly in molecular weight (57,000-59,000) and isoelectric point (4.58-4.63), but heterogeneity in their carbohydrate side-chains was suggested by concanavalin A-Sepharose 4B chromatography. The absorption spectra of the predominating forms 4 and 5 showed maxima of 278, 380, and 460 nm, indicative of flavoprotein character. Detailed comparative kinetic studies of forms 4 and 5 revealed few significant differences in behavior. Both proteins showed pH optima between 6.0 and 7.0, had identical Km values (0.17 mM) for (R,S)-mandelonitrile, and retained similar activities upon storage at 4 and -20 degrees C. Neither form exhibited a metal ion requirement and both were affected similarly by metal salts, beta-mercaptoethanol, and sulfhydryl reagents. Benzoic acid, p-hydroxybenzyl alcohol, and benzyl alcohol inhibited both forms.