The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric CO(2). A less appreciated function of CO(2) fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent CO(2) fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with (13)C-labeled acetate. R. palustris metabolized 22% of acetate provided to CO(2) and then fixed 68% of this CO(2) into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that CO(2) fixation plays a major role in cofactor recycling. When H(2) production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of CO(2) was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N(2) fixation and CO(2) fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO(2) fixation to recycle reduced cofactors deserves closer scrutiny.