The spatiotemporal properties of the Ca²⁺-release process in skeletal muscle fibers from normal and mdx fibers were determined using the confocal-spot detection technique. The Ca²⁺ indicator OGB-5N was used to record action potential-evoked fluorescence signals at consecutive locations separated by 200 nm along multiple sarcomeres of FDB fibers loaded with 10- and 30-mM EGTA. Three-dimensional reconstructions of fluorescence transients demonstrated the existence of microdomains of increased fluorescence around the Ca²⁺-release sites in both mouse strains. The Ca²⁺ microdomains in mdx fibers were regularly spaced along the fiber axis, displaying a distribution similar to that seen in normal fibers. Nevertheless, both preparations differed in that in 10-mM EGTA Ca²⁺ microdomains had smaller amplitudes and were wider in mdx fibers than in controls. In addition, Ca²⁺-dependent fluorescence transients recorded at selected locations within the sarcomere of mdx muscle fibers were not only smaller, but also slower than their counterparts in normal fibers. Notably, differences in the spatial features of the Ca²⁺ microdomains recorded in mdx and normal fibers, but not in the amplitude and kinetics of the Ca²⁺ transients, were eliminated in 30-mM EGTA. Our results consistently demonstrate that Ca²⁺-release flux calculated from release sites in mdx fibers is uniformly impaired with respect to those normal fibers. The Ca²⁺-release reduction is consistent with that previously measured using global detection techniques.