Cardiomyocytes are intrinsically fluorescent and spectroscopic analysis of rabbit ventricular myocytes indicated that the majority of this fluorescence arises from the metabolic coenzymes nicotinamide adenine dinucleotide (NADH) in the reduced state and flavin adenine dinucleotide (FAD) in the oxidised state. The mitochondrial redox state of single cells was assessed using the mitochondrial inhibitors p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) and cyanide. Treatment with 2μM FCCP established a completely oxidised state, resulting in NADH fluorescence decreasing to a minimum and FAD fluorescence increasing to a maximum. Treatment with cyanide (2mM) established a completely reduced cellular state, generating maximal NADH and minimal FAD fluorescence. The mitochondrial redox state of single rod-shaped myocytes was examined with confocal microscopy. NADH was excited at 405nm and FAD at 488nm. From this, the average NADH redox state was calculated as 0.58±0.03 (n=22) and the average redox state of FAD was calculated as 0.17±0.01 (n=22). Similar experiments using 2-photon excitation fluorescence microscopy (exciting at 720 and 750nm) revealed a comparable value for NADH redox state of 0.57±0.04 (n=20), although FAD fluorescence could not be clearly detected with this method. Measurements of intrinsic fluorescence were then utilised in order to assess the mitochondrial redox response of cardiac cells to increased energy demand. Isolated cardiomyocytes were field stimulated and fractional shortening simultaneously recorded with epifluorescence measurements of NADH and FAD. Cells were paced at 0.5Hz and the stimulation frequency step increased to 1Hz, 2Hz and 3Hz in order to increase work intensity and energy demand. NADH was excited at 340nm and FAD was excited at 430nm. Step increasing stimulation frequency resulted in a decrease in NADH fluorescence and an increase in FAD fluorescence, indicating oxidation of the cell environment. The magnitude of response was dependent on stimulation frequency. The greatest response was obtained when pacing was increased from 0.5Hz to 3Hz, NADH fluorescence decreased by 11.16±1.42% and FAD increased by 11.74±1.34% (n=22). Reducing work intensity back to 0.5Hz pacing resulted in immediate recovery of metabolite fluorescence. In conclusion, the majority of intrinsic fluorescence from isolated heart cells could be attributed to the metabolic coenzymes NADH and FAD. Metabolic inhibition enabled modulation of the oxidative status of these enzymes, allowing for calculation of relative redox state. Simultaneous measurements of redox status and fractional shortening in field-stimulated cells have demonstrated that rabbit ventricular myocytes become oxidised when work rates are increased, suggesting a transient mismatch between energy supply and demand.