Hydrogen sulfide (H2S) has been traditionally considered to be a toxic molecule for mammals. However, it can be formed endogenously and exert physiological effects with potential health benefits. H2S can partition two-fold in biological membranes and traverse them rapidly, diffusing between compartments. H2S reactivity has similarities to that of thiols, although it is less nucleophilic than thiols and it can form different products. H2S can react with oxidants derived from the partial reduction of oxygen, but direct scavenging is unlikely to explain H2S protective actions. Important effects are exerted on mitochondria including the stimulation or the inhibition of the electron transport chain. Possible mechanisms for unleashing biological consequences are the reactions with metal centers and with thiol oxidation products. The reactions of H2S with disulfides (RSSR) and sulfenic acids (RSOH) lead to the formation of persulfides (RSSH). Persulfides have enhanced nucleophilicity with respect to the corresponding thiol, consistent with the alpha effect. Besides, the inner and outer sulfurs can both act as electrophiles. In this review, we describe the reactions of H2S with oxidized thiol products and the properties of the persulfides formed in the context of the chemical biology of H2S.