The energy transduction by complex I from Rhodothermus marinus was addressed by studying the influence of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on the activities of this enzyme. EIPA is an inhibitor of both Na+/H+ antiporter and complex I NADH:quinone oxidoreductase activity. We performed studies of NADH:quinone oxidoreductase and H+ and Na+ translocation activities of complex I from R. marinus at different concentrations of EIPA, using inside-out membrane vesicles. We observed that the oxidoreductase activity and both H+ and Na+ transports are inhibited by EIPA. Most interestingly, the catalytic and the two transport activities showed different inhibition profiles. The transports are inhibited at concentrations of EIPA at which the catalytic activity is not affected. In this way the catalytic and transport activities were decoupled. Moreover, the inhibition of the catalytic activity was not influenced by the presence of Na+, whereas the transport of H+ showed different inhibition behaviors in the presence and absence of Na+. Taken together our observations indicate that complex I from R. marinus performs energy transduction by two different processes: proton pumping and Na+/H+ antiporting. The decoupling of the catalytic and transport activities suggests the involvement of an indirect coupling mechanism, possibly through conformational changes.