l-Cystathionine γ-lyase [EC 220.127.116.11] of Saccharomyces cerevisiae was shown to bind cofactor pyridoxal 5′-phosphate, up to 2 molecules/subunit. The association constants of the enzyme for the cofactor were estimated to be 3.67 × 105 M–1 and 9.05 × 103 M–1 . However, the latter value was too small for the binding to play a catalytic role. Changes in the absorption spectra of the enzyme in γ-elimination reaction mixtures with various amino acids as substrates were observed at 10°C to elucidate the reaction mechanism of the enzyme. The enzyme formed a chromophore exhibiting absorption at approximately 480 nm, which is characteristic of an aminocrotonate intermediate with O-succinyl-l-homoserine, l-cystathionine, l-homoserine, or O-acetyl-l-homoserine, at rates in this order. The intermediate was consumed at much lower rates than those of formation. The order of the rates of consumption was the same as the order of the formation rates and the order of the γ-elimination activity of the enzyme with the above-mentioned substrates. These results strongly suggested that the intermediate was essential for γ-elimination and that the reaction was rate-limited by its conversion into the product α-ketobutyrate. l-Cysteine sensitively inhibited the α, γ-elimination activity of the enzyme, and also retarded the formation of the chromophore when it was provided to the enzyme together with a substrate. The reason for these phenomena is discussed.