We used mice with genetic disruption of the A3 adenosine receptor (AR) gene (A3AR(-/-)mice) to assess the in vivo role of the A3AR in modulating myocardial ischemia/reperfusion injury and preconditioning (PC). Surprisingly, infarct size induced by 30 min of coronary artery occlusion and 24 h of reperfusion was 35% smaller in A3AR(-/-)compared to wild-type mice (A3AR(+/+)). The reduction in infarct size was not the result of differences in heart rate, body temperature or increased cardiac expression of A1ARs. However, neutrophil infiltration within infarcted regions was less in A3AR(-/-)mice. Furthermore, ischemic PC induced by either a single episode (one 5 min occlusion) or multiple episodes (six 4 min occlusions) of ischemia produced equivalent reductions in infarct size in A3AR(-/-)and A3AR(+/+)mice. These results indicate that, in the mouse, (i) A3ARs play an injurious role during acute myocardial ischemia/reperfusion injury, possibly by exacerbating the inflammatory response, and (ii) A3ARs are not necessary for the development of the early phase of ischemic PC.