In an attempt to determine whether the natural ATPase inhibitor (IF1) plays a role in oxidative phosphorylation, the time course of ATP synthesis and ATP hydrolysis in inside-out submitochondrial particles from beef heart mitochondria either possessing IF1 (Mg-ATP particles) or devoid of IF1 (AS particles) was investigated and compared to movements of IF1, as assessed by an isotopic assay. The responses of the above reactions to preincubation of the particles in aerobiosis with NADH or succinate were as follows: (1) The few seconds lag that preceded the steady-rate phase of ATP synthesis was shortened and even abolished both in Mg-ATP particles and AS particles. The rate of ATP synthesis in the steady state was independent of the length of the lag. (2) ATPase was slowly activated, maximal activation being obtained after a 50-min preincubation; there was no direct link between the development of the protonmotive force (maximal within 1 sec) and ATPase activation. (3) Bound IF1 was slowly released; the release of bound IF1 as a function of the preincubation period was parallel to the enhancement of ATPase activity; the maximal amount of IF1 released was a small fraction of the total IF, bound to the particles (less than 20%). (4) The double reciprocal plots of the rates of ATP and ITP hydrolysis vs. substrate concentrations that were curvilinear in the absence of preincubation with a respiratory substrate became linear after aerobic preincubation with the substrate. The data conclusively show that only ATPase activity in submitochondrial particles is correlated with the release of IF1, and that the total extent of IF1 release induced by respiration is limited. On the other hand, the kinetics of ATPase in control and activated particles are consistent with the existence of two conformations of the membrane-bound F1-ATPase, directed to ATP synthesis or ATP hydrolysis and distinguishable by their affinity for IF1.