Endogenously produced hydrogen sulfide is thought to function as an intracellular messenger. There is, however, little information on tissue concentrations of free hydrogen sulfide, the putative messenger form of this molecule, versus that of the bound (acid-labile) form. The present report describes the application of a novel technique to measure free and acid-labile hydrogen sulfide in mouse tissues. Very low free hydrogen sulfide concentrations (<0.050μmol/kg) were observed in brain, liver, blood, heart, kidney, striated muscle, and esophagus. Aortic concentrations of free hydrogen sulfide were 20 to 100 times greater than that of the other tissues. Acid-labile hydrogen sulfide concentrations were multiple orders of magnitude greater than that of the free form in every tissue other than aorta. Previous reports of tissue hydrogen sulfide concentrations of 30 to >100μmol/kg measured bound rather than free hydrogen sulfide, the observation that aorta contains anomalously high free hydrogen sulfide concentrations lends support for a vasodilator function for this molecule, and the very low free hydrogen sulfide concentrations in most tissues seemingly requires intermediation of a yet to be described receptor-like mechanism if this molecule is to serve as a gasotransmitter.