&NA;We investigated the roles of sodium‐calcium exchange, sarcoplasmic reticulum, and mitochondria in Caihomeostasis in cultured chick ventricular cells. Specifically, the influence of low sodium medium on contractile state, calcium fluxes, and cytosolic free [Ca] ([Ca]i) was examined. [Ca]iwas measured using fura‐2. Mean [Ca]iin control medium was 126 ± 14 nM. Exposure of cells to sodium‐free or sodium‐ and calcium‐free medium (choline‐substituted) resulted in contracture development, which returned toward the baseline level over 2‐3 minutes. The Nao‐free contracture was associated with a tenfold increase in [Ca]i(1,280 ± 110 nM) followed by a gradual decrease to a level fourfold above control [Ca]i(460 ± 58 nM). Nao‐ and Cao‐free contracture was associated with a fivefold increase in [Ca]i(540 ± 52 nM) followed by a rapid decrease to below 80 nM. Sodium‐free medium failed to produce an increase in [Ca]ior contracture in cells preexposed to calcium‐free medium, although caffeine, when subsequently added to sodium‐ and calcium‐free medium, was able to elicit a transient increase in [Ca]iand contracture. Brief, 5‐second preperfusion of cells with La3+(1 mM) or EGTA (1 mM) abolished the Nao‐free contracture and the increase in [Ca]i. In the presence of 20 mM caffeine, removal of Naoresulted in minimal changes in the resting position of the cell although45Ca uptake and [Ca]iwere increased in response to sodium‐free medium; the subsequent decrease in [Ca]iwas greatly slowed. Addition of caffeine during the relaxation phase of the sodium‐free contracture produced an additional transient contracture and transient increase in [Ca]i. Ryanodine (1 μM) abolished this effect of caffeine. Caffeine or ryanodine abolished Nao‐ and Ca‐free contracture. CCCP (2 μM), a potent oxidative phosphorylation inhibitor, did not significantly affect calcium efflux rate. In the presence of 2 μM CCCP, removal of sodium resulted in an augmented contracture signal and a rise in [Ca]i, followed by a slow decrease. We conclude that removal of extracellular sodium enhances transsarcolemmal entry of calcium via sodium‐calcium exchange, but this effect alone does not lead to the development of sodium‐free contracture. Calcium displaceable by lanthanum or EGTA appears to contribute to Nao‐free or Nao‐ and Cao‐free contracture. Studies using caffeine and ryanodine suggest that removal of Naoleads to release of calcium from the sarcoplasmic reticulum (presumably via calcium‐induced calcium release). The release calcium appears to be taken up by both mitochondria and sarcoplasmic reticulum and is in part extruded by the ATP‐dependent sarcolemmal calcium pump, causing spontaneous relaxation. (Circulation Research1987;61:29‐41)