Strenuous activitiy of skeletal muscle leads to temporary osmotic dysbalance and isolated skeletal muscle fibers exposed to osmotic stress respond with characteristic micro-domain calcium signals. It has been suggested that osmotic stress targets transverse tubular (TT) dihydropyridine receptors (DHPRs) which normally serve as voltage-dependent activators of Ca release via ryanodine receptor (RyR1s) of the sarcoplasmic reticulum (SR). Here, we pursued this hypothesis by imaging the response to hyperosmotic solutions in both mouse skeletal muscle fibers and myotubes. Ca fluctuations in the cell periphery of fibers exposed to osmotic stress were accompanied by a substantial dilation of the peripheral TT. The Ca signals were completely inhibited by a conditioning depolarization that inactivates the DHPR. Dysgenic myotubes, lacking the DHP-receptor-alpha1-subunit, showed strongly reduced, yet not completely inhibited activity when stimulated with solutions of elevated tonicity. The results point to a modulatory, even though not essential, role of the DHP receptor for osmotic stress-induced Ca signals in skeletal muscle.