Stretch activates an array of signalling pathways in the heart, regulating the force and rhythm of contraction and gene expression. A role for caveolae, invaginated lipid rafts lined with caveolin, in sensing and transducing mechanical stimuli has been proposed, but direct evidence for this is lacking in the adult cardiac myocyte. Here we investigate the caveolar localisation of 3 proteins linked with the contractile (eNOS, NHE) and electrical (TREK1) response of the heart to stretch, and determine the time-dependent effect of stretch on the distribution of caveolin 3 (Cav3), eNOS, NHE and TREK1. Left ventricular (LV) pressure (measured via a balloon in the LV attached to a pressure transducer) and LV monophasic action potentials (MAPs) were recorded in Langendorff-perfused adult rat hearts. Some hearts were stretched by inflating the balloon to give 95% of maximum developed pressure; inflation was maintained for 10 or 30 min. To obtain caveolae-enriched membranes, LVs were fractionated on a discontinuous sucrose gradient following detergent-free Na₂CO₃ extraction. Stretch caused both an immediate and a secondary slow increase in developed pressure (P<0.05 vs. pre-stretch or immediately post-stretch respectively; paired Student’s t-test; n=14 hearts). MAP duration at 75% and 90% repolarisation was prolonged immediately after stretch (P<0.05). Buoyant caveolae-containing fractions (BF; 4-6 of 12) were enriched in Cav3 but excluded the non-caveolar marker β-adaptin, which was found predominantly in heavy fractions (HF; 9-12). In the absence of stretch, 100% of eNOS and NHE1 was found in BFs, whereas 96 ± 4% of TREK-1 was outside BFs (mean ± S.E.M; n=7). Stretch caused a translocation of Cav 3 from caveolae; at 30 min after stretch the ratio of Cav 3 in BF/HF was reduced from 1.6 ± 0.2 to 1.1 ± 0.1 (P<0.05, t-test; n=7). For the mechanotransductive proteins, the relationship between % in BF and time of stretch showed a tendency for eNOS (R=-0.93) to move from caveolae and for TREK1 (R=+0.91) to move to caveolae. NHE1 distribution did not change with stretch (R=-0.11). In conclusion, some proteins (eNOS, NHE) involved in mechanotransduction in the adult heart are located exclusively in caveolae. Our data illustrate the dynamic nature of caveolae in response to mechanical stimuli. Stretch causes progressive movement of Cav3 from caveolae, consistent with disruption of caveolar structure, and translocation of eNOS from, and TREK1 to, caveolae. Because caveolae can modify signalling by concentrating or excluding elements of signal transduction cascades, and because Cav3 itself can interact to regulate protein activity, translocation of proteins to/from caveolae lends weight to the hypothesis that these microdomains are involved in mechanotransduction in the heart.