The Pi ⇄ HOH exchange reaction of oxidative phosphorylation is considerably less sensitive to uncouplers than the Pi ⇄ ATP and ATP ⇄ HOH exchanges. The uncoupler-insensitive Pi ⇄ HOH exchange is inhibited by oligomycin. These results and other considerations suggest that the relatively rapid and uncoupler-insensitive Pi ⇄ HOH exchange results from a rapid, reversible hydrolysis of a tightly but noncovalently bound ATP at a catalytic site for oxidative phosphorylation, concomitant with interchange of medium and bound Pi. Such tightly bound ATP has been demonstrated in submitochondrial particles in the presence of uncouplers, Pi, and ADP, by rapid labeling from 32Pi under essentially steady-state phosphorylation conditions. These results lead to the working hypothesis that in oxidative phosphorylation energy from electron transport causes release of preformed ATP from the catalytic site. This release could logically involve energy-requiring protein conformational change.