The mechanism of inhibition of yeast F0F1-ATPase by its naturally occurring protein inhibitor (IF1) was investi-gated in submitochondrial particles by studying the IF1-mediatedATPase inhibition in the presence andabsence of a protonmotive force. In the presence of protonmotive force, IF1 added during net NTP hydrolysis almost completely inhibitedNTPase activity. At moderate IF1 concentration, subsequent uncoupler addition unexpect-edly caused a burst of NTP hydrolysis.