Aims: Hydrogen sulfide (H 2 S) is a signaling molecule, which influences many physiological processes. While H 2 S is produced and degraded in many cell types, the kinetics of its turnover in different tissues has not been reported. In this study, we have assessed the rates of H 2 S production in murine liver, kidney, and brain homogenates at pH 7.4, 37°C, and at physiologically relevant cysteine concentrations. We have also studied the kinetics of H 2 S clearance by liver, kidney, and brain homogenates under aerobic and anaerobic conditions. Results: We find that the rate of H 2 S production by these tissue homogenates is considerably higher than background rates observed in the absence of exogenous substrates. An exponential decay of H 2 S with time is observed and, as expected, is significantly faster under aerobic conditions. The half-life for H 2 S under aerobic conditions is 2.0, 2.8, and 10.0 min with liver, kidney, and brain homogenate, respectively. Western-blot analysis of the sulfur dioxygenase, ETHE1, involved in H 2 S catabolism, demonstrates higher steady-state protein levels in liver and kidney versus brain. Innovation: By combining experimental and simulation approaches, we demonstrate high rates of tissue H 2 S turnover and provide estimates of steady-state H 2 S levels. Conclusion: Our study reveals that tissues maintain a high metabolic flux of sulfur through H 2 S, providing a rationale for how H 2 S levels can be rapidly regulated. Antioxid. Redox Signal. 17, 22-31.