Toxicosis by monensin, a Na+-selective ionophore, induces skeletal and cardiac muscle necrosis. Cultured neonatal rat cardiac myocytes were killed by monensin (>0.2 μg/ml) beginning at 30 min and completing by 60-90 min. Other cultured cell types presumably lacking excitable membranes were not killed by monensin under these conditions. Cardiac myocytes were also killed by nigericin and nonactin (monovalent cation-carrying ionophores with low ion selectivity), but not by valinomycin, which has a high selectivity for K+. Monensin-induced killing was associated with formation of blebs in cell membranes and subsequent swelling of the cells during the early phases of killing, whereas surface membranes of cells permeabilized to trypan blue dye contained discrete small holes visible by scanning electron microscopy. Monensin-induced killing occurred at extracellular Na+ concentrations ≥10 mm, but not when Li+, K+, Cs+, Rb+, or choline ions replaced Na+ at concentrations up to 0.15 m. Killing was prevented at extracellular pH values ≤6.4 and was enhanced by ouabain, an inhibitor of Na+ K+-ATPase-mediated Na+ transport. Several characteristics of monensin-induced cardiac myocyte killing were similar to those observed during killing induced by the Ca2+-carrying ionophore, A23187 plus Ca2+, including a requirement for extracellular Ca2+ concentrations >0.5 mm, inhibition by Mn2+ and Ni2+, and an associated stimulation of arachidonic acid release. The cell killing characteristics are consistent with a monensin-induced Na+ influx which admits toxic levels of extracellular Ca2+ to the cytoplasm of cells with excitable membranes, possibly via Na+ Ca2+ antiporter protein(s).