We used mice with genetic disruption of the A₃adenosine 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 inA₃AR^−/− 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.