Cystathionine β-lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5´-phosphate-dependent β-cleavage of cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding cystathionine β-lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure. Physicochemical properties of the Arabidopsis cystathionine β-lyase were similar to those previously reported for the bacterial enzymes. In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5´-phosphate. Interaction between the apoenzyme and pyridoxal 5´-phosphate is extremely tight, being characterized by a Kd value of 0.5 µM. Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278. The substrate specificity of the recombinant cystathionine β-lyase resembles that of the enzyme isolated from other sources, cystathionine and djenkolate being the most effective substrates. The cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant cystathionine β-lyase. The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5´-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine.