The aims of this study were to determine: (i) whether clonazepam and CGP37157, which inhibit the Na+/Ca2+ exchanger of isolated mitochondria, could inhibit mitochondrial Ca2+ efflux in intact cells; and (ii) whether any sustained increase in mitochondrial [Ca2+] ([Ca2+](m)) could alter mitochondrial NADH levels. [Ca2+](m) was measured in Indo-1/AM loaded rat ventricular myocytes where the cytosolic fluorescence signal was quenched by superfusion with Mn2+. NADH levels were determined from cell autofluorescence. Upon exposure of myocytes to 50 nM norepinephrine (NE) and a stimulation rate of 3 Hz, [Ca2+](m) increased from 59 ± 3 nM to a peak of 517 ± 115 nM (n = 8) which recovered rapidly upon return to low stimulation rate (0.2 Hz) and washout of NE. In the presence of clonazepam, the peak increase in [Ca2+(m) was 937 ± 192 nM (n = 5) which remained elevated at 652 ± 131 nM upon removal of the stimulus. CGP37157 in some cells did give the same inhibition of mitochondrial Ca2+ efflux as clonazepam, but the effect was inconsistent since not all cells were capable of following the stimulation rate in presence of this compound. NADH levels increased upon exposure to rapid stimulation in the presence of NE alone and recovered upon return to low stimulation rates, whereas in clonazepam treated cells the recovery of NADH was prevented. We conclude that clonazepam is an effective inhibitor of mitochondrial [Ca2+] efflux in intact cells and also maintains the increase in NADH levels which occurs upon rapid stimulation of cells.