The cardiac Na/Ca exchanger's (NCX1) role in calcium homeostasis during myocardial contractility makes it a possible target of signaling factors regulating inotropy. Caveolae, structured invaginations of the plasmalemma, are known to concentrate a wide variety of signaling factors. The predominant coat proteins of caveolae, caveolins, dock to and regulate the activity of these signaling factors and other proteins through interaction with their scaffolding domain. In this study we investigated the interaction of NCX1 with caveolin proteins. Western blots of bovine cardiac sarcolemmal vesicles revealed the presence of caveolin-1, -2, and -3. Immunoprecipitation of detergent-solubilized vesicle proteins with either NCX1 or caveolin-3 antibodies indicated that NCX1 coprecipitates with caveolin-3, but not with caveolin-1 and -2. Functional disruption of caveolae, by beta-cyclodextrin treatment of vesicles, diminished coprecipitation of caveolin-3 and NCX1 activity. NCX1 has five potential caveolin-binding motifs, two of which are in the transporter's exchange inhibitory peptide (XIP) domain. The presence of 50 mM XIP peptide enhanced coprecipitation of caveolin-3 with NCX1 independent of calcium concentration. We conclude that NCX1 associates specifically with caveolin-3. Partitioning of NCX1 in caveolae has implications for temporal and spatial regulation of excitation-contraction and -relaxation coupling in cardiac myocytes.