The amplitude of coronary reactive hyperemia (CRH), elicited by 15 s of ischemia, is reduced in hearts subjected to 5 min of ischemic preconditioning (IP). F0F1 ATP synthase activity and ATP concentration are also altered by IP. We hypothesized that F0F1 ATP synthase is differently modulated by the inhibitor protein IF(1) during CRH elicited before (CRHnp) and after (CRHprec) IP. Hemodynamic parameters were recorded in 10 anesthetized goats. Myocardial biopsies were obtained before IP (Cnp), during CRHnp, 4 and 6 min after the onset of CRHnp, after IP (Cprec), during CRHprec, and 4 min after CRHprec. F0F1 ATP synthase activity, ATP concentration, and ATP-to-ADP ratio (ATP/ADP) were determined. Compared with CRHnp, IP blunted CRHprec. F0F1 ATP synthase activity transiently increased during CRHnp, decreased 4 min after CRHnp, and returned to control 2 min later; it was lower after IP (Cprec) and did not change during and after CRHprec. All these changes in activity were modulated by IF1. During CRHnp, ATP concentration and ATP/ADP were reduced compared with Cnp and began to rise 6 min thereafter. During Cprec, both parameters were transiently reduced but increased during and after CRHprec. Hence, during CRHnp, F0F1 ATP synthase activity transiently increases and then decreases significantly. The short-lasting inhibition of the enzyme may explain why a few seconds of occlusion do not induce IP. After IP, F0F1 ATP synthase activity is blunted, and it is not affected by a subsequent 15 s of occlusion, which induces a blunted CRHprec. These results suggest that postischemic long-lasting inhibition of F0F1 ATP synthase activity may be a feature of the preconditioned heart. The increase in ATP concentration after preconditioning is in agreement with previous reports of reduced ATP hydrolysis by cytoplasmic ATPases.