β-(6-Benzylaminopurin-9-yl)alanine (3), a metabolite of the cytokinin 6-benzylaminopurine (2), was confirmed to be derived from 0-acetyl-L-serine (1) and 2 by an enzyme in higher plants. The enzyme preparations were mainly obtained from immature seeds and seedlings of Lupinus luteus. The formation of 3 was demonstrated through incubation of reaction mixtures containing 1, 2, and enzyme preparations, followed by chromatographic analysis (HPLC, paper chromatography) and identification with an automatic amino acid analyzer. Some properties of the enzyme were described: it was specific for 1 as the alanyl-moiety donor (no activity with O-acetyl-D-serine, O-phospho-L-serine, or L-serine); the optimal pH for enzymatic formation of 3 was 8.0-8.3; the Km value for 1 was 4.5 mM; low concentration of pyridoxal 5'-phosphate (0.23 mM) increased the reaction rate by about 25% while high concentration (2.3 mM) inhibited it by 15%; the enzyme activity was inhibited by NH2OH (50 mM) or KCN (2 mM) by more than 50%. Enzyme activity for 3 formation was detected in seedlings of L. luteus and Pisum sativum, and fresh leaves of Spinacia oleracea, with the highest activity in immature seeds of L. luteus. It was suggested that enzymes catalyzing β-substituted alanine formation showed differences in specificity for heterocyclic substrates among different plant species. Further investigation on the enzymes is in progress.