More than 15 years have passed since hydrogen sulfide (H2S) emerged as a biological signaling molecule – initially, in the nervous and vascular systems through the modulation of N-methyl d-aspartate (NMDA) receptors and the activation of transmembrane receptor potential (TRP)- and ATP-dependent K+ (KATP)-channels (Abe and Kimura, 1996; Hosoki et al., 1997; Dello Russo et al., 2000; Zhao et al., 2001; Teague et al., 2002; Nagai et al., 2004; Streng et al., 2008), and later, in nearly every organ system (Predmore and Lefer, 2010). H2S was found to play a role as a cytoprotectant in the nervous system (Kimura and Kimura, 2004; Whiteman et al., 2004); this finding led to the discovery of the cardioprotective effect of H2S (Elrod et al., 2007) (Figure ​(Figure1).1). Previous studies have also described that H2S plays several roles: as a regulator of insulin release, inflammation, and angiogenesis, and as an oxygen (O2) sensor (Li et al., 2005; Yang et al., 2005; Kaneko et al., 2006; Olson et al., 2006; Zanardo et al., 2006; Cai et al., 2007; Papapetropoulos et al., 2009). Figure 1 Production, metabolism, storage, and functions of H2S. Previous studies report measurement of endogenous concentrations of sulfide by methods involving high concentrations of acids; therefore, contamination by free H2S released from acid-labile sulfur resulted in an overestimate of the free H2S levels (50–160 μM; Goodwin et al., 1989; Warenycia et al., 1989; Savage and Gould, 1990). A sulfur/silver electrode has frequently been used for the measurement of sulfide concentrations in biological samples. The electrode measures the level of S2−, and a pKa value of 13.9 results in the replacement of cysteine sulfide groups in proteins with hydroxyl groups, thereby releasing H2S from proteins. Because tissue and blood samples contain abundant proteins, this method estimates erroneously high concentrations of sulfide (Whitfield et al., 2008). Recently, the basal or steady state endogenous concentrations of H2S have been re-evaluated using methods that avoid release of contaminant H2S from proteins; these methods give concentration estimates of 20 nM to a few micromolar in tissue and blood samples (Furne et al., 2008; Ishigami et al., 2009; Wintner et al., 2010). It is necessary to determine the active state concentrations of H2S. At least three factors influence H2S concentration: (1) rate of H2S production, (2) rate of H2S metabolism, and (3) storage of H2S as bound sulfane sulfur and its associated release.