Endogenous H₂S was recognized recently as a new gaseous signaling molecule which plays beneficial roles in vasodilation and facilitation of long-term potentiation. The interaction of mammalian myoglobin (Mb) with H₂S occurs via two distinct pathways: the first is the direct binding of H₂S to the heme-Fe center of the Mb to form the Mb-H₂S adduct, and the second is the covalent modification with H₂S at a porphyrin vinyl group of the Mb heme to form the covalently modified sulfheme. We chose Mb as the prototype heme-containing protein and probed the interactions of different Mb mutants with H₂S by UV-vis spectroscopy, and X-ray crystallography. The heme active sites of Mb mutants directly affect the binding of H₂S to the proteins and alsothe formation of sulfheme derivatives. This provides critical experimental data for elucidating how heme-containing proteins modify the metabolism of endogenous H₂S.