To identify the actual substrate of the glutathione-dependent sulfur dioxygenase (EC 220.127.116.11) elemental sulfur oxidn. of the meso-acidophilic Acidithiobacillus thiooxidans strains DSM 504 and K6, Acidithiobacillus ferrooxidans strain R1 and Acidiphilium acidophilum DSM 700 was analyzed. Extraordinarily high specific sulfur dioxygenase activities up to 460 nmol min-1 (mg protein)-1 were found in crude exts. All cell-free systems oxidized elemental sulfur only via glutathione persulfide (GSSH), a non-enzymic reaction product from glutathione (GSH) and elemental sulfur. Thus, GSH plays a catalytic role in elemental sulfur activation, but is not consumed during enzymic sulfane sulfur oxidn. Sulfite is the first product of sulfur dioxygenase activity; it further reacted non-enzymically to sulfate, thiosulfate or glutathione S-sulfonate (GSSO-3). Free sulfide was not oxidized by the sulfur dioxygenase. Persulfide as sulfur donor could not be replaced by other sulfane-sulfur-contg. compds. (thiosulfate, polythionates, bisorganyl-polysulfanes or monoarylthiosulfonates). The oxidn. of H2S by the dioxygenase required GSSG, i.e. the disulfide of GSH, which reacted non-enzymically with sulfide to give GSSH prior to enzymic oxidn. On the basis of these results and previous findings a biochem. model for elemental sulfur and sulfide oxidn. in Acidithiobacillus and Acidiphilium spp. is proposed.