The relationships among intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i, respectively) and cell morphology were investigated during metabolic inhibition (MI) in isolated guinea pig myocytes. [Na+]i and [Ca2+]i were measured using the fluorescent indicators, Na(+)-binding benzofuran isophthalate and fluo 3. During the initial 20 min of MI, [Na+]i increased from 6.2 +/- 0.5 to 18.6 +/- 1.6 mM (n = 31), whereas [Ca2+]i, expressed as the percent change of fluo 3 fluorescence, remained at the low level. In the following 30 min, 94% of the cells developed contracture, and [Ca2+]i began to increase after cells had contracted (167 +/- 14% at 50 min). The level of [Ca2+]i during MI was lower than that during 500 microM strophanthidin perfusion. The increase in [Na+]i was not affected by 10 microM tetrodotoxin but was suppressed by 1 microM hexamethylene amiloride (HMA). The application of 10 mM glucose from the start of MI prevented both the increase in [Na+]i and cell contracture. However, the addition of glucose after 20 min of MI [energy repletion (ER)] led to a dramatic increase in [Ca2+]i (442 +/- 72% at 50 min, n = 31), and 84% of the cells developed contracture. The increase in [Ca2+]i and the cell contracture were suppressed by HMA or Ca(2+)-free solution. Intracellular pH decreased from 7.23 +/- 0.07 to 6.95 +/- 0.09 during MI but did not change after ER (6.90 +/- 0.11 at 35 min, n = 9). These findings suggested that during MI 1) [Na+]i increased by both the activated Na+ influx via Na+/H+ exchange and the suppressed Na+ extrusion via the Na+/K+ pump, 2) Na+/Ca2+ exchange was inhibited by energy depletion and intracellular acidosis, and 3) cell contracture was not related to Ca2+ overload but was related to rigor due to energy depletion.