The enzymes of the transsulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are important for the endogenous production of hydrogen sulfide (H(2)S), a gaseous signaling molecule. The relative contributions of CBS and CSE to H(2)S generation in different tissues are not known. In this study, we report quantification of CBS and CSE in murine liver and kidney and their contribution to H(2)S generation in these tissues and in brain at saturating substrate concentrations. We show that CBS protein levels are significantly lower than those of CSE; 60-fold and 20-fold in liver and kidney, respectively. Each enzyme is more abundant in liver compared with kidney, twofold and sixfold for CBS and CSE, respectively. At high substrate concentrations (20 mM each cysteine and homocysteine), the capacity for liver H(2)S production is approximately equal for CBS and CSE, whereas in kidney and brain, CBS constitutes the major source of H(2)S, accounting for ∼80% and ∼95%, respectively, of the total output. At physiologically relevant concentrations of substrate, and adjusting for the differences in CBS versus CSE levels, we estimate that CBS accounts for only 3% of H(2)S production by the transsulfuration pathway enzymes in liver.