Cystathionine beta-lyase, the second enzyme involved in the methionine biosynthetic pathway in plants, catalyses the synthesis of homocysteine from cystathionine. A cDNA encoding cystathionine beta-lyase was cloned from an Arabidopsis thaliana expression library by complementation of an Escherichia coli mutant deficient in this enzyme. As deduced from the full-length nucleotide sequence (1.7 kb), the polypeptide contains 464 amino acids and presents a predicted M(r) of 50372. A. thaliana cystathionine beta-lyase exhibits 22% sequence identity with the E. coli corresponding enzyme and contains a 70 amino acid N-terminal additional sequence compared with the bacterial protein. Since the general features of chloroplast transit peptides could be observed in this amino-terminal extension, we propose a chloroplast localization for the cDNA-encoded enzyme. Southern blot analysis suggested that cystathionine beta-lyase is encoded by a single copy gene in A. thaliana.