The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD(+) +NADH) to the vesicles caused a similar to 4-fold or similar to 1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD(+) +NADH both were added. The change was lacking in the presence of the F0F1-ATPase inhibitors N,N'-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F0F1-ATPase and, in addition, it was less in potassium ion-free medium. These results are correlated with data about K+-dependent F0F1-ATPase activity, suggesting a relationship between the F0F1-ATPase and K+ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.