Activity of dark assimilation of carbon dioxide by marine heterotrophic thiosulfate-oxidizing bacteria increased in the presence of thiosulfate by as much as 500% depending on the rate of thiosulfate oxidation. Pyruvate produced a similar effect on dark CO2 assimilation. Activity of dark assimilation in the sulfate-forming group of heterotrophic bacteria was about one order higher than in tetrathionate-forming bacteria and in heterotrophic bacteria incapable of thiosulfate oxidation. Dark assimilation of carbon dioxide in marine heterotrophic thiosulfate-oxidizing bacteria, which are unable to grow autotrophically, is however presumed to play an important role in biomass synthesis via utilization of energy derived from thiosulfate oxidation in the process of energy-consuming pyruvate carboxylation.