The aim of the present study was to determine whether the attenuation of myocardial ischemic injury by SB203580 is due to the inhibition of p38 mitogen-activated protein kinase (MAPK) or to other documented nonspecific effects of the drug. We made adenoviral vectors encoding the &agr; isoform of p38 MAPK with or without site-directed mutations to prevent SB203580 binding and inhibition. In embryonal rat heart–derived cells and adult rat cardiocytes expressing wild-type p38&agr; MAPK, injury was reduced significantly by SB203580 present during simulated ischemia. In contrast, SB203580 did not protect cells expressing the SB203580-resistant form of p38&agr; MAPK. These observations suggest that SB203580-mediated protection depends on the inhibition of p38&agr; MAPK.