In this study we measured the exogenous production and the intracellular content of elemental sulfur (S0) in the thermophilic sulfur-oxidizing bacteria Hydrogenobacter spp. and Bacillus schlegelii during mixolithoautotrophic growth on hydrogen and thiosulfate. Under these conditions, all strains produced and released white-yellow hydrophilic S0 particles into the growth medium. Hydrophilic S0 was separated from cells by a differential low-speed centrifugation procedure. The S0 pellets were dried, and the S0 was purified by column chromatography and by thin-layer chromatography (TLC). The S0 TLC-band could be stained with triphenyltetrazolium chloride and piperidine procedure. Determination of intracellular S0 content was performed from fresh cells absolutely free of exogenous S0 particles. Quantitation of S0 was performed by high-performance liquid chromatography, colorimetric thiocyanate procedure, and by UV-spectra analyses. All the strains studied, in particular B. schlegelii strains, released significant quantities of S0 into the growth media. In contrast, the intracellular S0 content was very low. Significant rhodanese activity in the presence of thiosulfate was measured with toluene-permeabilized cells and cell-free extracts of B. schlegelii (type strain) and Hydrogenobacter spp. (strain T3).