1. Intracellular calcium concentration [( Ca2+]i) and pH (pHi) were measured in single, isolated rat ventricular myocytes using, respectively, the fluorescent indicators Fura‐2 and BCECF (2',7'‐bis(carboxyethyl‐5(6)‐carboxyfluorescein). Contraction was measured simultaneously. The intracellular calibration of BCECF is demonstrated. In a HEPES‐buffered bathing solution of pH 7.4, pHi had a mean value of 7.16 +/‐ 0.05 (mean +/‐ S.E.M.). 2. Addition of NH4Cl (5‐20 mM) produced an intracellular alkalosis that was associated with an increase of contraction amplitude. Removal of NH4Cl produced an acidosis and decrease of contraction. 3. The addition of 2 mM‐cyanide (CN‐) to inhibit oxidative phosphorylation had variable effects on contraction amplitude. Changes of contraction amplitude could largely be accounted for by changes in the systolic Ca2+ transient. 4. CN‐ addition increased lactic acid production. However, in the majority of experiments, this was not accompanied by an intracellular acidosis. 5. Anaerobic glycolysis was inhibited by either removal of glucose, addition of deoxyglucose, or addition of iodoacetate. Under these conditions the application of CN‐ decreased systolic [Ca2+]i and contraction amplitude. This was sometimes preceded by a transient increase of systolic [Ca2+]i and contraction amplitude. 6. When glycolysis was inhibited, the subsequent addition of CN‐ always increased diastolic [Ca2+]i and produced a contracture. The increase of [Ca2+]i occurred before the contracture. However, once the contracture had developed, decreasing [Ca2+]i (by removal of external Ca2+) did not cause relaxation. 7. With glycolysis inhibited, addition of CN‐ resulted in a large (0.51 +/‐ 0.05 pH unit) acidosis that was sometimes preceded by an alkalosis. This acidosis was unaffected by removal of external Ca2+ or external alkalinization. Calculations show that some of this acidosis may result from protons released by ATP hydrolysis. 8. If the acidosis produced by metabolic blockade was partly reversed by adding NH4Cl then a contracture immediately developed. This suggests that the acidosis delays the onset of the contracture. 9. We conclude that metabolic inhibition increases diastolic [Ca2+]i. The accompanying acidosis prevents contraction. Once the contracture has developed it is maintained by factors other than increased [Ca2+]i, possibly by a fall of [ATP].