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The gene encoding human cystathionine γ-lyase was cloned from total cellular Hep G2 RNA. Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian cystathionine γ-lyase overproduced in a bacterial system. About 90% of the heterologous gene product was insoluble, and renaturation experiments from purified inclusion bodies met with limited success. About 5 mg/liter culture of human cystathionine γ-lyase could also be extracted from the soluble lysis fraction, employing a three-step native procedure. While the enzyme showed high γ-lyase activity toward l-cystathionine (K _m = 0.5 mm,V _max = 2.5 units/mg) with an optimum pH of 8.2, no residual cystathionine β-lyase behavior and only marginal reactivity toward l-cystine and l-cysteine were detected. Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and …