Elevated free Ca²⁺ concentrations found in adult dystrophic muscle fibers result in enhanced protein degradation. Since the difference in concentrations may reflect differences in entry, Ca²⁺ leak channels in cultures of normal and Duchenne human myotubes, and normal and mdx murine myotubes, have been identified and characterized. The open probability of leak channels is markedly increased in dystrophic myotubes. Other channel properties, such as mean open times, single channel conductance, ion selectivity, and behavior in the presence of pharmacological agents, were similar among myotube types. Compared to the Ca²⁺ concentrations in normal human and normal mouse myotubes, intracellular resting free Ca²⁺ concentrations ([Ca²⁺]_i) in myotubes of Duchenne and mdx origin were significantly higher at a time when dystrophin is first expressed in normal tissue. Taken together, these findings suggest that the increased open probability of Ca²⁺ leak channels contributes to the elevated free intracellular Ca²⁺ concentration in Duchenne human and mdx mouse myotubes.