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Cytoplasmic ATP can be measured continuously in single cardiac myocytes by monitoring the luminescence from microinjected firefly luciferase. We show here that the signals are markedly influenced by changes in cytoplasmic pH, and the calibration of the signals as ATP concentration is markedly affected by cytoplasmic protein. Measurements with a pH-sensitive fluorescent dye show that intracellular pH (pHi) can be clamped at pH 7.08 by perfusing cells with a modified bicarbonate-buffered Krebs saline containing 92 mM NaHCO3 and equilibrated with 20% CO2. Calibration of the firefly luciferase signal in vitro in the presence of high concentrations of BSA (180 mg/ml), to simulate the cytoplasmic protein concentration, revealed a shift in Km (ATP) to 2 mM, from approx. 400 microM in the absence of albumin in an identical ionic milieu. Luciferase measurements in pH-clamped cells show that metabolically poisoned isolated rat ventricle cardiomyocytes enter rigor at a cytoplasmic ATP concentration of between 1 and 2 mM. As the cells shorten in rigor, a process that is complete in 30-40 s, the cytoplasmic ATP concentration falls simultaneously to a level of typically 20 microM. When cyanide is removed 10 min later, to simulate reoxygenation, the signal recovers over a period of 2-3 min to a level approx. 70% of the original in the healthy cell. These studies indicate that rigor-mediated depletion of cytoplasmic ATP in metabolically poisoned cardiomyocytes is considerably more extreme than hitherto indicated.