The hypothesis that calcium influxes from the extracellular space play an important role in the pathogenesis of irreversible anoxic injury was tested using isolated adult rat myocytes. Myocytes treated with 6 mm amytal and 3 mm iodoacetate and subsequently incubated in either calcium-containing (1.12 mm) or calcium-free media (with or without 1 mm EGTA) developed rigor contracture (cell squaring) and cell death (trypan blue permeability) at the same rate. The rates of cell death in both calcium-containing and calcium-free media were increased by incubation in hypotonic media even though the rates of contracture development remained unaltered. Cells developed osmotic fragility prior to membrane permeability increases. The calcium ionophore, A23187 (10 μm), induced rapid rounding of rod-shaped cells subjected only to mitochondrial inhibition in calcium containing media, confirming its ability to cause an increase in cellular permeability to calcium. However, A23187 did not alter the rates of cell death of totally metabolically inhibited myocytes in either calcium-containing or calcium-free media with EGTA. The results indicate that influxes of calcium are not necessary for the development of irreversible injury in metabolically inhibited, isolated myocytes.