CASK (Ca2+, calmodulin-associated serine/threonine kinase) is an essential mammalian cell junction protein and is also crucial at Drosophila neuromuscular synapses. We have shown that CASK is present in mammalian skeletal muscle at the postsynaptic membrane of the neuromuscular junction. CASK interacts biochemically with channels at central synapses, and studies in cultured cells have led to proposed functions for CASK. However, in vivo functions of CASK in skeletal muscle remain unknown. To test hypotheses of CASK functions, we generated two lines of transgenic mice, which overexpress full-length and truncated CASK protein in skeletal muscle. Extensive analyses showed that overexpression of CASK protein did not affect the morphology or physiology of skeletal muscle, the morphology of the neuromuscular junction, or the levels or distribution of protein interactors. These results contrast with previous cell culture experiments and emphasize the importance of in vivo analysis of protein function.