1. Single, intact muscle fibres were dissociated enzymatically from skeletal muscles of phenotypically normal (+/?) and dystrophic mice (129/ReJ dy/dy: Dystrophia muscularis), and resting Ca2+ levels were measured by image analysis of intracellular Fura-2 fluorescence in distinct parts of the fibres. 2. Fura-2 was introduced into fibres by ionophoresis with glass microelectrodes to concentrations of between 50 and 200 microM. Over this concentration range there was no apparent buffering of intracellular Ca2+ by Fura-2. 3. Fibres isolated from the soleus, flexor digitorum brevis (FDB) and extensor digitorum longus (EDL) muscles of normal animals maintained resting [Ca2+] of 106 +/- 2 nM. Ca2+ distributions within individual fibres were homogeneous. 4. Fibres from dystrophic animals maintained [Ca2+] that was elevated two- to fourfold in comparison to normal fibres. 5. The population of skeletal fibres from dystrophic mice which displayed morphology similar to that of fibres of normal animals were found to have Ca2+ levels that averaged 189 +/- 2 nM. The distribution of Ca2+ within these fibres appeared uniform. 6. The population of dystrophic fibres that possessed morphological abnormalities maintained even higher Ca2+ concentrations (368 +/- 3 nM). Several fibres from this morphological group displayed obvious heterogeneity in Ca2+ distribution with distinct, localized areas of higher Ca2+. 7. These results support the contention that Ca2+ homeostasis is markedly impaired in dystrophic muscle. The elevated Ca2+ levels are near the threshold for contraction and, together with severe morphological fibre abnormalities, are probably centrally involved in fibre necrosis apparent in muscular dystrophy.