Intracellular staining for calcium was studied in muscle biopsies from 15 dogs by the alizarin red S (ARS) stain. Rare positive fibres were present in normal muscle and in denervation atrophy. The percentage of positive fibres was slightly increased in polymyositis, dermatomyositis and canine temporal/masseter myositis and markedly increased in progressive muscular dystrophy. Calcium-positive fibres were usually so-called large-dark (hypercontracted) fibres or necrotic fibres, although there was occasional staining of normal and atrophied fibres. These results indicate the probable involvement of calcium in muscle injury in canine inflammatory myopathies and in canine muscular dystrophy. In addition, use of the ARS stain appears to be useful for detecting the earliest lesions of acute muscle fibre injury.