In cultured embryonic chick heart cells, alterations of extracellular Na (Nao) and Ca (Cao), intracellular Na (Nai) and Ca, extracellular pH, and membrane potential resulted in changes in Na and Ca contents that were consistent with sarcolemmal Na-Ca exchange. 24Na efflux measurements revealed a large ouabain-insensitive component, one-third of which was inhibited by removal of Cao. Incubating the cells in Na-free solution resulted in a rapid, 1.5- to 2-fold increase in total cell Ca that remained elevated for at least 15 min. Cells exposed for 15 min to Nao less than or equal to 20 mM became maximally loaded with Ca, whereas Ca loading fell off sharply at values of Nao greater than 20 mM. The movement of Na against its electrochemical gradient was shown to be associated with Ca accumulation. During Na-K pump inhibition (in 10(-4) M ouabain), Na initially rose 2- to 3-fold to a level below its equilibrium value; then, lowering Cao for 30 min from 1.25 to 0.75 mM caused a 26% elevation in Nai, whereas raising Cao from 1.25 to 2.7 mM resulted in a 25% fall in Nai against its electrochemical gradient. These data are consistent with Nai being maintained by a Na-Ca exchange during Na-K pump inhibition. In the presence of ouabain (10(-4) M), Ca uptake into intracellular organelles, e.g., mitochondria, was suggested by an increase in total cell Ca as well as the occurrence of mitochondrial matrix granules, which were shown qualitatively by X-ray analysis to contain Ca. Although matrix granules also occurred in mitochondria during Na-free incubation, they did not contain detectable amounts of Ca when examined under identical conditions of fixation and analysis.