We studied the calcium compartmentation of the Ca overload induced by 50 min of metabolic inhibition (MI) in cultured neonatal rat cardiomyocytes. MI was achieved by application of 1 mM iodoacetic acid (IAA) and 10 mM 2-deoxyglucose (2-DOG) with omission of glucose in the perfusate. MI per se abolished spontaneous beating, but 10 mM caffeine pulses at 15, 30 and 45 min were able to induce contractile responses. The contractile responses were abolished in the presence of Thg. After 20 min of MI, cells began to accumulate Ca. After 50 min of MI, the Ca exchanged via Na/Ca exchange (Na/Ca exchange-dependent Ca content) was increased by 3.21 +/- 0.15 mmol/kg dry weight (n = 5). The net increase remaining exchangeable via Na/Ca exchange represents 80% of the global net 45Ca uptake after 50 min in MI which was 3.98 mmol/kg dry weight, obtained in the "on-line" experiments. The release of this component of Ca is completed within 150s via Na/Ca exchange. There also was a 50% increase in the Ca content of the so-called "slow" compartment, 0.44 +/- 0.08 mmol/kg dry weight (n = 10) after 50 min under MI. The t1/2 of this compartment was 24.8 +/- 3.8 min typical of a mitochondrial origin in these cells. Thus 92% of the net Ca increase after 50 min MI remains exchangeable-80% of this is completed within 150s via Na/Ca exchange and 20% more slowly, t1/2 about 25 min. Cell ATP content, 27.35 +/- 2.07 nmoles/mg protein, was decreased after 10 min of MI to 2.25 +/- 0.29 nmoles/mg protein (n = 10) and remained close to this level after 20 min of MI (2.11 +/- 0.17 nmoles/mg protein; n = 6). It is to be noted that our results indicate no compromise of the Na/Ca exchange system after 50 min of MI although the global ATP is depleted in these cells.