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Hydrogen sulfide has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious and neuropathological diseases have been undertaken. Here we review the possible direct targets of H₂S in mammals. H₂S directly interacts with reactive oxygen/nitrogen species and is involved in redox signaling. H₂S also reacts with hemeproteins and modulates metal-containing complexes. Once being oxidized, H₂S can persulfidate proteins by adding -SSH to the amino acid cysteine. These direct modifications by H₂S have significant impact on cell structure and many cellular functions, such as tight junctions, autophagy, apoptosis, vesicle trafficking, cell signaling, epigenetics and inflammasomes. Therefore, we conclude that H₂S is involved in many important cellular and physiological processes. Compounds that donate H₂S to biological systems can be developed as therapeutics for different diseases.