Caveolae, small invaginations in the plasma membrane, contain caveolins (Cav) that scaffold signaling molecules including the tyrosine kinase Src. We tested the hypothesis that cardiac protection involves a caveolin-dependent mechanism. We used in vitro and in vivo models of ischemia-reperfusion injury, electron microscopy (EM), transgenic mice, and biochemical assays to address this hypothesis. We found that Cav-1 mRNA and protein were expressed in mouse adult cardiac myocytes (ACM). The volatile anesthetic, isoflurane, protected ACM from hypoxia-induced cell death and increased sarcolemmal caveolae. Hearts of wild-type (WT) mice showed rapid phosphorylation of Src and Cav-1 after isoflurane and ischemic preconditioning. The Src inhibitor PP2 reduced phosphorylation of Src (Y416) and Cav-1 in the heart and abolished isoflurane-induced cardiac protection in WT mice. Infarct size (percent area at risk) was reduced by isoflurane in WT (30.5+/-4 vs. 44.2+/-3, n=7, P<0.05) but not Cav-1(-/-) mice (46.6+/-5 vs. 41.7+/-3, n=7). Cav-1(-/-) mice exposed to isoflurane showed significant alterations in Src phosphorylation and recruitment of C-terminal Src kinase, a negative regulator of Src, when compared to WT mice. The results indicate that isoflurane modifies cardiac myocyte sarcolemmal membrane structure and composition and that activation of Src and phosphorylation of Cav-1 contribute to cardiac protection. Accordingly, therapies targeted to post-translational modification of Src and Cav-1 may provide a novel approach for such protection.