Cytochrome aa3 concentrations in the cytoplasmic membrane of Bacillus subtilis were altered by growth conditions, and the effects on the membrane potential (delta psi) in whole cells were measured. When cytochrome aa3 was absent, the magnitude of delta psi was not diminished by comparison with the delta psi measured in cells containing normal cytochrome aa3 concentrations. In addition, the energy-dependent uptake of proline and glutamate was comparable at both cytochrome aa3 concentrations. However, in the cytochrome aa3-deficient cell preparation, accumulation of the aminoglycoside antibiotic streptomycin was much lower than that of the cytochrome aa3-sufficient cells. When cells were cultured under conditions that stimulated higher than normal concentrations of cytochrome aa3, delta psi was also increased, and enhanced streptomycin accumulation was observed. Phenazine methosulfate-ascorbate was used both in delta psi measurements and in uptake studies to provide high rates of electron transport and maximal delta psi values. These results, taken together with those previously published (A. S. McEnroe and H. W. Taber, Antimicrob. Agents Chemother. 26:507-512, 1984) suggest that the uptake of streptomycin by B. subtilis requires adequate levels both of delta psi and cytochrome aa3.