The effects of phosphocreatine (PCr) on sarcoplasmic reticulum (SR) Ca(2+) regulation were investigated in saponin-permeabilized rat ventricular myocytes. Cells were perfused continuously with weakly Ca(2+)-buffered solutions approximating to the intracellular milieu. Ca(2+) release from the SR was detected using Fura-2 or Fluo-3. Withdrawal of PCr reduced the frequency of spontaneous Ca(2+) release by 12.8 +/- 3.4 % (n = 9) and the amplitude of the spontaneous Ca(2+) transient by 17.4 +/- 3.1 % (n = 9). Stepwise reductions in [PCr] progressively increased the time for the spontaneous Ca(2+) transient to rise from 25 to 100 % of the maximum value (TP75) and to fall by 75 % of the peak level (DT75). Following complete PCr withdrawal, the TP75 and the DT75 were 147.1 +/- 13.2 and 174.8 +/- 23.2 % of the control values, respectively. Experiments involving confocal microscopy showed that PCr withdrawal decreased the propagation velocity of spontaneous Ca(2+) waves. PCr withdrawal also reduced the frequency and amplitude, but increased the duration of spontaneous Ca(2+) sparks. Rapid application of 20 mM caffeine was used to assess the SR Ca(2+) content at the point of spontaneous Ca(2+) release. In the absence of PCr, the amplitude of the caffeine-induced Ca(2+) transient was 18.4 +/- 2.7 % (n = 9) lower than in the presence of 10 mM PCr. This suggests that PCr withdrawal reduces the maximum SR Ca(2+) content that can be sustained before spontaneous Ca(2+) release occurs. These results suggest that local ADP buffering by PCr is essential for normal Ca(2+) regulation by the SR. Prolongation of the descending phase of the spontaneous Ca(2+) transient is consistent with a reduction in the efficiency of the SR Ca(2+) pump due to ADP accumulation. The fact that spontaneous Ca(2+) release occurs at a lower SR Ca(2+) content in the absence of PCr suggests that the Ca(2+) release mechanism may also be affected. These effects may be of relevance in circumstances where PCr depletion and Ca(2+) overload occur, such as myocardial ischaemia or anoxia.