Mechanisms of energy deprivation contracture were investigated in cultured chick embryo ventricular cells. In the presence of zero-extracellular-Na+(choline chloride substitution)-nominal-zero-Ca2+([Ca2+] ˜ 5 μM), exposure of ventricular cells to 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG)-zero-glucose solution resulted in the development of a contracture (video motion detector) in 5.9 ± 0.5 minutes. Early after contracture development, the resupply of extracellular Na+, in the continued presence of CN + 2-DG, resulted in a rapid partial relaxation (tl/2= 1.9 ± 0.3 seconds), associated with an increase in45Ca efflux, presumably due to transsarcolemmal Ca2+extrusion due to Na+-Ca2+exchange. Ressupply of glucose and removal of CN + 2-DG, in the continued absence of Na+, resulted in an initially slower (t1/2= 11.6 ± 2.5 seconds), but more complete relaxation of contracture, which was not associated with increased Ca2+efflux. Pretreatment with 20 mM caffeine delayed the onset of contracture (9.2 ± 1.1 minutes) and resulted in a contracture that could not be relaxed by ressupply of external Na+only. Studies using the fluorescent Ca2+probe indol demonstrated that in zero-Na+-zero-Ca2+solutions, contracture due to CN + 2-DG was associated with an initial rise in [Ca2+]1but that this did not account for all of contracture force development. In cells exposed to CN + 2-DG in the presence of normal extracellular Na+and Ca2+concentrations, a small rise in [Ca2+]1was associated with initial contracture development, consistently preceding the development of a larger accelerated contracture presumably due to ATP depletion. We conclude that an early component of ATP depletion contracture is due to an increase in [Ca2+]1. The rate of this increase in [Ca2+]1depends to some extent on the loading of internal stores of Ca2+, particularly sarcoplasmic reticulum. Elevation of [Ca2+], may promote subsequent rigor by hastening ATP depletion by activation of Ca2+-ATPases. (Circulation Research 1987;61:726-734)