The ejection of protons from oxygen-pulsed cells and the gradients of Na + concentration ( Na o + Na i + at 150 mM external NaCl) and proton electrochemical potential (Δ μ H + ~) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F 0F 1-ATPase inhibitors dicyclohexylcarbodiimide (F 0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F 1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 ± 0.15 to 0.7 ± 0.1 nmol/ mg dry wt while overall O 2 uptake and Δ μ H + ~ were much less affected. H + efflux was inhibited only 60 to 75%. Aerobic Na o + Na i + ratios (5.9 ± 0.6) under these conditions remained 50% above the anaerobic level (2.1 ± 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H + efflux and O 2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H + O ratios from 4.0 ± 0.3 to 1.9 ± 0.2. Similarly, with KCN and CO the Na o + Na i + ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 m m KCN. Measurements of the proton motive force (Δ μ H + ~) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na + H + antiporter for the active exclusion of Na + from the cell interior.