Uncertainty exists regarding the mechanisms of Ca efflux from isolated myocardial cells. Therefore, transmembrane fluxes of Ca, exchangeable Ca content, and contractile behavior were studied in monolayers of cultured chick embryo-ventricle during abrupt exposure to media containing zero [Na]o (choline chloride substitution). Exposure to zero [Na]o induced a transient contracture of cultured heart cells. Rapid Ca influx increased significantly over the first 60 s immersion in zero [Na]o; however, total exchangeable Ca content, measured by labelling with 45Ca, did not change, suggesting that rapid Ca efflux was also increasing under these conditions. When 45Ca in the extracellular space was removed by washing for 16 s in ice cold buffer before 45Ca efflux was measured, an increase in Ca efflux with exposure to zero [Na]o was apparent. Treatment of cells with 10(-3) M cyanide and 20 mM 2-deoxyglucose resulted in a decrease in the Ca efflux in zero [Na]o-zero [Ca]o medium, and in the appearance of significant components of [Na]o and [Ca]o-dependent Ca efflux. These results are consistent with the hypothesis that Ca efflux in these cells normally occurs predominantly by a non-[Na]o-dependent mechanism, probably an ATP-dependent Ca pump. After metabolic blockade of ATP production with a resulting increase in Ca loading of the cytoplasm, increased efflux of Ca via Na Ca and Ca Ca exchange occurs.