The tissue and subcellular distribution in liver and kidney of bound and acid-labile sulfur as well as the enzyme capacity for sulfide production related to the desulfuration pathway were determined in rats. Bound sulfur was widely distributed in tissues and highest in kidney, whereas acid-labile sulfur was highest in heart. Bound sulfur was found primarily in the cytosolic fraction in the form of high molecular weight material in liver, and as both high and low molecular weight material in kidney. Acid-labile sulfur was located in the mitochondrical fraction. Sulfide production capacity from cysteine was greatest in liver cytosol. This capacity was well correlated with the distribution of gamma-cystathionase in tissues and subcellular fractions.