Muscle cells from Duchenne muscular dystrophy (DMD) patients and the dystrophic mdx mouse lack the protein dystrophin. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myofibers and cultured myotubes and is correlated with abnormally active calcium-specific leak channels. Higher [Ca2+]i results in greater calcium-dependent proteolysis, which may eventually lead to necrosis. We performed liposome-mediated transfection of a cDNA plasmid containing the full-length dystrophin gene into mdx myoblasts and examined the resting [Ca2+]i and leak channel activity of the resulting differentiated myotubes. Many myotubes from transfected cultures expressed dystrophin at levels similar to normal myotubes as determined by immunostaining. The intracellular free calcium, measured by emission ratio microfluorimetry using the calcium indicator fura-PE3, was significantly lower in the dystrophin-positive mdx myotubes than in untransfected control mdx myotubes. The mean open probability of the calcium leak channel was also reduced to a level similar to normal myotubes and significantly less than that for untransfected mdx myotubes. These results show that introduction of extrachromosomal copies of the full dystrophin gene to originally dystrophic muscle cells can correct the defect in calcium homeostasis that is hypothesized to lead to the muscle cell necrosis seen in DMD.