It has recently been shown that various stress-inducing manipulations in isolated ventricular myocytes may lead to significant remodelling of t-tubules. Osmotic stress is one of the most common complications in various experimental and clinical settings. This study was therefore designed to determine the effects of a physiologically relevant type of osmotic stress, hyposmotic challenge, to the integrity of the t-tubular system in mouse ventricular myocytes using the following two approaches: (1) electrophysiological measurements of membrane capacitance and inward rectifier (IK1) tail currents originating from K(+) accumulation in t-tubules; and (2) confocal microscopy of fluorescent dextrans trapped in sealed t-tubules. Importantly, we found that removal of '0.6 Na' (60% NaCl) hyposmotic solution, but not its application to myocytes, led to a ∼27% reduction in membrane capacitance, a ∼2.5-fold reduction in the amplitude of the IK1 tail current and a ∼2-fold reduction in the so-called IK1 'inactivation' (due to depletion of t-tubular K(+)) at negative membrane potentials; all these data were consistent with significant detubulation. Confocal imaging experiments also demonstrated that extracellularly applied dextrans become trapped in sealed t-tubules only upon removal of hyposmotic solutions, i.e. during the shrinking phase, but not during the initial swelling period. In light of these data, relevant previous studies, including those on excitation-contraction coupling phenomena during hyposmotic stress, may need to be reinterpreted, and the experimental design of future experiments should take into account the novel findings.