In this study we investigated whether NADH fluorescence levels changed in response to low or high rates of electrical stimulation in single ventricular myocytes isolated from rat and guinea-pig hearts, either during a single contraction or upon sustained electrical stimulation of cells. NADH levels were determined from cell autofluorescence and cell length monitored using an edge-tracking device. NADH/NAD + was obtained by addition of cyanide, 100% NADH, and carbonylcyanide- p-trifluoromethoxy phenylhydrazone (FCCP), 100% NAD +. Rat myocytes exhibited slightly higher resting fluorescence levels than guinea-pig cells; however, NADH/NAD + was higher in rat than guinea-pig cells ( P < 0.05), 24.3 ± 4.3 (N = 17) vs 14.6 ± 1.6 (N = 17), respectively. There was no change in NADH fluorescence during a single contraction when cells were stimulated at either low (0.2 Hz) or high (3 Hz) rates in either species. Furthermore, NADH levels did not change upon sustained stimulation at 3 Hz in either species. Metabolic blockade with cyanide induced a dose dependent rise in NADH fluorescence which was similar for both rat and guinea-pig myocytes and reached a maximum at ≥ 1 mM of cyanide. Although a full recovery of NADH fluorescence was seen in both types of cells after brief exposure to cyanide, the rate of recovery was significantly slower in rat myocytes; times to 90% recovery were 110 ± 29 sec, N = 6, and 264 ± 50 sec, N = 6, for guinea-pig and rat cells, respectively. This work demonstrates that although rat and guinea-pig myocytes have different resting NADH/NAD +, their response to electrical stimulation is the same, whereas in response to metabolic inhibition subtle differences are seen.