By use of 31P NMR, the transmembrane pH gradient (ΔpH) and the intracellular levels of phosphorylated metabolites were measured in aerobic suspensions of wild-type Escherichia coli cells in the presence and absence of the adenosinetriphosphatase (ATPase) inhibitor dicyclohexylcarbodiimide (DCCD); the same parameters were also determined in E. coli mutants deficient in ATPase activity under both anaerobic and aerobic conditions. A method is described by which dense suspensions of E. coli cells (∼3 × 1011 cells/mL) were oxygenated so that steady-state O2 levels in the suspensions were far greater than the Km for O2 consumption. Under these conditions, in wild-type MRE600 cells, the intracellular concentrations of Pi, NTP, and NDP were measured to be 3.0 ± 1.5, 8 ± 1, and 1.2 ± 1 mM, respectively, while the intracellular pH was ∼7.5 over the external pH range studied (6 to ∼7.0). Upon treatment with DCCD, the intracellular NTP level was drastically reduced and intracellular Pi concentration increased in respiring wild-type cells; in the same cells, however, DCCD did not affect the intracellular pH and the ApH. During respiration in the presence of lactate, ATPase− cells established a ApH but failed to synthesize any detectable levels of NTP. Conversely, ATPase− cells accumulated high levels of NTP but did not generate a ApH during glycolysis under anaerobic conditions. These results are in complete agreement with the generally accepted chemiosmotic hypothesis. 31P NMR data on intact ATPase− NR70 cells were in agreement with the previously proposed [Rosen, B.P., Brey, R., & Hasan, S. (1978) J. Bacteriol. 134, 1030] existence of a proton leak in this strain which is sealed by DCCD or by spontaneous mutation into strain NR71. However, the NMR data also indicated that other major differences exist between NR71 and NR70 cells.