Insulin induces the expression of Hsp70 in rat heart. In this study we examined insulin and heat shock treated hearts for improved contractile recovery after 30 minutes of ischemia, activation of the heat shock transcription factor and for localization of the Hsp70 in relation to dystrophin and -tubulin. Adult male Sprague-Dawley rats were assigned to groups: 1) control, 2) sham control, 3) insulin injected (200 µUnits/gm body weight), 4) heat shock treated (core body temperature 42 °C for 15 min), 5) heat shock and insulin treated. Six hours later, hearts were isolated for Langendorff perfusion to determine cardiac function, or myocardial tissues were collected and prepared for either electrophoretic-mobility shift assay, Western blot analysis or immunofluorescence microscopy. Insulin treatment with 6 hrs of recovery enhances post-ischemic myocardial recovery of contractile function and increases Hsp70 expression through activation of the heat shock transcription factor. Insulin treated hearts had elevated levels of Hsp70 particularly in the membrane fraction. In contrast, heat shocked hearts had elevated levels of Hsp70 in the cytosol, membrane and pellet fractions. After insulin treatment Hsp70 was mostly co-localized to the plasma membrane with dystrophin. In contrast, after heat shock, Hsp70 was localized mostly between cardiomyocytes in apparent vascular or peri-vascular elements. The localization of Hsp70 is dependent on the inducing stimuli of either heat shock or insulin treatment. The cell membrane versus vascular localization of Hsp70 suggests the interesting possibility of functionally distinct roles for Hsp70 in the heart, whether induced by insulin or heat shock treatment.