The hypothesis that intracellular Ca2+ is elevated in dystrophic (mdx) skeletal muscle due to increased Ca2+ influx is controversial. As the sub-sarcolemmal Ca2+ ([Ca2+](mem)) should be even higher than the global cytosolic Ca2+ in the presence of increased Ca2+ influx, we investigated [Ca2+](mem) levels in collagenase-isolated adult flexor digitorum brevis (FDB) myofibres and myotubes of mdx and normal mice with the near-membrane Ca2+ indicatorFFP-18. Confocal imaging showed strong localization of FFP-18 to the sarcolemma only. No significant difference in [Ca2+](mem) was found in FDB myofibres of normal (77.3 +/- 3.8 nM, n = 68) and mdx (79.3 +/- 5.6 nM, n = 21, p = 0.89) mice using FFP-18. Increasing external Ca2+ to 18 mM did not significantly affect [Ca2+](mem) in either the normal or mdx myofibres. In the myotubes, the FFP-18 was non-selectively incorporated, distributing throughout the cytoplasm, and FFP-18-derived [Ca2+] values were similar to values obtained with Fura-2. Nevertheless, in the mdx myotubes, the [Ca2+] measured with FFP-18 increased linearly to a level similar to 2.75 times that of controls as the time of culture was prolonged. In older mdx myotubes (>= 8 days in culture), 18 mM extracellular Ca2+ increased the steady state cytosolic [Ca2+] to similar to 22 times greater level than controls. This study suggests that the sub-sarcolemmal Ca2+ homeostasis is well maintained in isolated adult mdx myofibers and also further supports the hypothesis that cytosolic Ca2+ handling is compromised in mdx myotubes. (c) 2006 Elsevier Ltd. All rights reserved.