The efficiency factor, the number of moles of ATP generated per mole of glucose fermented, was determined in anaerobic, non-carbon-limited N2-fixing cultures of Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and Clostridium butyricum through identification and quantitation of the fermentation products by 13C nuclear magnetic resonance spectroscopy and measurement of acetate kinase activities. All three Bacillus species had acetate kinase activities and produced acetate and ethanol as the major fermentation products. The maximum amounts of ATP generated per mole of glucose fermented were 2.70, 2.64, and 2.88 mol in B. polymyxa, B. macerans, and B. azotofixans, respectively, compared with 3.25 mol in C. butyricum. Thus, in the N2-fixing Bacillus species, the efficiency factors are lower than that in C. butyricum. Steady-state ATP/ADP concentration ratios were measured in non-carbon-limited N2-fixing cultures of B. polymyxa and B. azotofixans through separation and quantitation of the adenylates in cell extracts by ion-pair reversed-phase high-performance liquid chromatography. The observed ATP/ADP ratios were 4.5 and 3.8, and estimated energy charges were 0.81 to 0.86 and 0.81 to 0.83, respectively, for B. polymyxa and B. azotofixans. The results suggest that under these growth conditions, the rate of ATP regeneration is adequate to meet the energy requirement for N2 fixation in the Bacillus species, in contrast to N2-fixing Clostridium pasteurianum and Klebsiella pneumoniae, for which substantially lower steady-state ATP/ADP ratios and energy charges have been reported. Implications of the results are discussed in relation to possible differences between Bacillus and Clostridium species in energy requirements for N2 fixation and concomitant ammonia assimilation.