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Cardiac T-tubules present an extensive network of invaginations of the plasma membrane that conduct inside the myocyte the electrical signal for activation of intracellular Ca2+ release and contraction. Progressive T-tubule remodeling has been linked to compromised myocardial contractile function in chronic cardiac disease, including heart failure. However, the effects of acute stress such as interstitial edema that commonly observed during myocardial infarction on the integrity of the cardiac T-system have not been examined. In the present study we investigated the impact of experimental myocardial edema on the structural integrity of the T-tubule system in intact Langenorff-perfused rat hearts. For visualization of T-tubules the hearts were loaded with the membrane-staining fluorescent dye di-8-ANEPPS and fluorescin-dextran and cells in the pericardium layer were imaged with a confocal microscope. Myocardial interstitial edema (IE) was induced by increasing the osmolarity of the perfusion solution with mannitol (56 mM/L). We found that IE resulted in a marked disruption of t-tubule structure, including vacuolization. To further evaluate the loss of structural continuity of the T-tubules caused by IE we performed experiments in which after initial loading, fluorescin-dextran, was washed out in IE and control hearts. IE significantly slowed and reduced the extent of washout of fluorescin-dextran from the myocardium. Significant t-tubule staining remained in IE hearts even after 20 mins of washout when nearly all staining was lost in control hearts. These results suggest that acute myocardial edema causes marked disruption of the continuity and structural integrity of the cardiac T-tubule system. Such T-tubule disarrangement could contribute to contractile dysfunction during acute myocardial disease associated with edema including myocardial infarction.