1. We have investigated the mechanisms responsible for the changes of systolic Ca2+ that occur in voltage-clamped rat ventricular myocytes during acidosis produced by application of the weak acid butyrate (30 mm). Intracellular pH regulation was inhibited with dimethylamiloride (bicarbonate-free solution). 2. The application of butyrate produced an intracellular acidification of 0.33 pH units. This was accompanied by a decrease in systolic Ca2+ to about 50% of control. However, within 2 min, systolic Ca2+ returned to control levels. 3. The decrease in systolic Ca2+ was accompanied by a decrease in the Na+-Ca2+ exchange current observed on repolarisation so that the calculated Ca2+ efflux on Na+-Ca2+ exchange was less than the entry on the L-type Ca2+ current. The magnitude of the Na+-Ca2+ exchange current recovered along with systolic Ca2+ until it equalled the Ca2+ entry on the L-type Ca2+ current. 4. From the measurement of Ca2+ fluxes, it was calculated that, during acidosis, the cell gains 121.6 ± 16.2 μmol 1-1 of Ca2+. This is equal to the measured increase of sarcoplasmic reticulum (SR) calcium content obtained by applying caffeine (20 mm) and integrating the resulting Na+-Ca2+ exchange current. 5. We conclude that the recovery of the amplitude of the systolic Ca2+ transient is due to decreased SR calcium release, resulting in reduced Ca2+ efflux from the cell leading to increased SR calcium content.