Apoptosis is an active form of cellular death, or suicide, which plays an important physiologic role during organ development and in cellular turnover in differentiated tissues. Apoptosis has also been demonstrated to occur in several organs in response to hypoxic/ischemic, oxidative, or drug-induced injury and is thus involved in disease pathogenesis. However, it is generally assumed that apoptosis does not occur in differentiated skeletal muscle. Apoptosis has been demonstrated in differentiated myocardial muscle, neonatal skeletal muscle, and skeletal myoblasts in response to injury. We therefore studied differentiated murine C2 skeletal muscle cells that have been injured by supraphysiologic doses (>10 microM) of an anabolic steroid, stanozolol. Stanozolol-injured muscle cells exhibited pathologic features suggestive of apoptosis: cytoplasmic shrinkage and chromatin condensation. Muscle cells also showed positive in situ nick-end labeling of nuclear chromatin, indicating DNA strand breakage. Staining with the DNA-binding dye 33342 (bisbenzimide) also showed chromatin changes characteristic of apoptotic nuclei. Total protein levels measured at 4 and 24 hr post-stanozolol injury was not significantly decreased, indicating absence of cell lysis. Cellular ATP levels (nmol ATP/mg protein) of stanozolol-injured muscle cells, measured 4 and 24 hr postinjury, also did not change significantly. In contrast, necrotic muscle cells, injured by the calcium ionophore A23187 (2 microM), showed a progressive decline in total protein and ATP levels. This study supports two other histologic studies that showed evidence of apoptosis in differentiated skeletal muscle fibers. Our data further suggest that during the early stages of apoptosis, but not necrosis, cellular energy metabolism is preserved.