H₂S derived from thiol metabolism has been proposed serve as an oxygen sensor in many systems due to its susceptibility for oxidation and ability to mimic hypoxic responses in oxygen sensing tissues. Thiosulfate, an intermediate in oxidative H₂S metabolism can be reduced and regenerate H₂S. We propose this contributes to the H₂S-mediated oxygen sensing mechanism. H₂S formation from thiosulfate in buffers and variety of mammalian tissues and lamprey aorta was examined in real-time using a polarographic H₂S sensor. Inferences of intracellular H₂S production were made by examining hypoxic pulmonary vasoconstriction (HPV) in bovine pulmonary arteries under conditions expected to increase H₂S production and in mouse and rat aortas where reducing conditions mediate vasorelaxation. In mammalian and lamprey buffers, H₂S was generated from thiosulfate in the presence of the exogenous reductant, dithiothreitol (DTT), or the endogenous reductant dihydrolipoic acid (DHLA). Both magnitude and rate of H₂S production was greatly increased by these reductants in the presence of tissue with the most notable effects occurring in the liver. H₂S production was only observed when tissues were hypoxic; exposure to room air, or injecting oxygen inhibited H₂S production and resulted in net H₂S consumption. DTT and DHLA augmented HPV and DHLA dose-dependently relaxed precontracted mouse and rat aortas. These results indicate that thiosulfate can contribute to H₂S signaling under hypoxic conditions and that this is a ready source of H₂S production and it serves as a means of recycling sulfur thereby conserving biologically relevant thiols.