The amplitude of coronary reactive hyperemia (CRH), elicited by 15-seconds of ischemia, is reduced in hearts preconditioned by 5-minutes of ischemia (IP). The activity of F₀F₁ATP-synthase and ATP concentration are also altered by IP. We hypothesized that F₀F₁ATP-synthase is differently modulated by the inhibitor protein IF₁ during a CRH elicited before (CRHnp) and after (CRHprec) IP. Hemodynamic parameters were recorded in 10 anesthetized goats. Myocardial biopsies were taken before IP (Cnp), during CRHnp, 4 and 6 min after the onset of CRHnp, as well as after IP (Cprec), during CRHprec and 4 min after CRHprec. F₀F₁ATP-synthase activity, ATP concentration and the ATP/ADP ratio were determined. When compared with CRHnp, IP blunted the CRHprec. F₀F₁ATP-synthase activity transiently increased during CRHnp, decreased 4-min after CRHnp and returned to control value 2-min later. The activity was lower after IP (Cprec) and did not change during and after CRHprec. All these changes in activity were modulated by the inhibitor protein IF₁. During CRHnp, ATP concentration and ATP/ADP ratio were reduced compared with Cnp, and began rising 6 min after. During Cprec both parameters were transiently reduced, but increased during and after CRHprec. Hence, during a CRHnp, F₀F₁ATP-synthase activity transiently increases and then decreases significantly. The short-lasting inhibition of the enzyme may explain why few-seconds of occlusion do not induce IP. After IP, however, F₀F₁ATP-synthase activity is blunted and it is not affected by a subsequent 15-s occlusion, which induces a blunted CRHprec. These results suggest that postischemic long-lasting inhibition of F₀F₁ATP-synthase activity may be a feature of preconditioned heart. The increase in ATP concentration after preconditioning is in agreement with previous reports of a reduced ATP hydrolysis by cytoplasmic ATPases.