The effects of aeration during bacterial growth on the proton translocating activity of the respiratory chain of B. stearothermophilus ATCC 8005, which is stable enough for measurement of the H+/O ratio by an oxygen pulse method, were examined. For endogenous and ascorbate-N,N,N′,N′-tetramethyl p-phenylene diamine (TMPD) respiration, H+/O ratios of around 6 and 2 were obtained using resting cells grown under highly aerated conditions. The values were about 4 and 0 when cells were grown under limited-air conditions. Spectrophotometric and enzyme kinetical analyses revealed that both cytochrome caa3 and pigment-432 (cytochrome cao) were acting as terminal oxidases, while cytochrome b-558 (corresponding to the “cytochrome o-type oxidase” of the thermophilic bacterium PS3 in the previous paper [Sone, N., Kutoh, E., & Sato, K. (1990) J. Biochem, 107, 597–602]) was mainly serving in the cells grown under limited-air conditions. Measurement of the pH change upon ferrocytochrome c pulse with proteoliposomes reconstituted from the membrane extract of vigorously aerated cells and that of limited-air cells suggested that both cytochrome caa3 and pigment-432 (cytochrome cao) pump protons, while cytochrome b-558 does not.