The role of nitric oxide (NO) in redox cell signaling is widely accepted. However, the biological role of another candidate small inorganic signaling molecule and the subject of this study, hydrogen sulfide (H2S), is much less known. H2S as a reductant and nucleophile has numerous potential cellular targets; however, its rapid biological oxidation suggests a fleeting cellular existence. The challenge of accurate real-time measurement of H2S at low micromolar or nanomolar concentrations in biological preparations represents a major impediment to H2S investigations. We here demonstrate the use of a novel polarographic H2S sensor (PHSS) to follow rapid changes in H2S concentration in common buffered biological solutions with a detection limit near 10 nM. The PHSS, used in combination with O2 and NO sensors in multisensor respirometry, shows stability, a high signal-to-noise ratio, and signal specificity for H2S. Preparations of rat vascular tissue exhibit H2S production on the addition of sulfhydryl-bearing amino acid substrates and H2S consumption when supplied with exogenous H2S. Taken together, these findings suggest the existence of dynamic steady-state cellular H2S levels. The PHSS should facilitate the investigation of H2S biology by providing a previously unattainable continuous record of H2S under biologically relevant conditions.