The present studies provide new data on the involvement of menaquinol oxidases in substrate oxidation and energy conservation in aerobically grown, resting cells of Staphylococcus aureus 17810R, starved of endogenous energy reserves and supplemented with glutamate or L-lactate. These cells were energetically competent, since they oxidized both substrates, generated an electrochemical proton gradient (deltamuH+) and synthesized ATP via oxidative phosphorylation. Studies with KCN showed that: (i) L-lactate oxidation occurred via two terminal menaquinol oxidases - the ba3-type sensitive to low KCN and the bo-type insensitive to cyanide, (ii) glutamate oxidation proceeded via the bo-type oxidase, and (iii) ATP synthesis with glutamate or L-lactate was coupled only to the bo-type oxidase. Also in glucose-grown cells oxidizing L-lactate, ATP synthesis was coupled to the highly repressed bo-type oxidase. It is suggested that in the respiratory chain of strain 17810R two energy coupling sites may be present: in the complex of NADH-menaquinone oxidoreductase and in the complex of the bo-type menaquinol oxidase. The rate of ATP synthesis was similar with both substrates, but the rate of their oxidation differed significantly: the P/O ratios were 1.5 and 0.03 with glutamate and L-lactate, respectively. CCCP accelerated glutamate oxidation by 50% but was without effect on L-lactate oxidation. In cell lysates, the rates of NADH and L-lactate oxidation were equal. It is concluded that in whole cells of S. aureus 17810R oxidation of NADH derived from glutamate breakdown is tightly coupled to phosphorylation, while L-lactate oxidation seems to be rather loosely coupled.