1. The aim of this study was to investigate the effects of 20 mM extracellular lactate on Ca2+ regulation mechanisms in enzymatically isolated single guinea-pig cardiac myocytes. 2. The activities of the Ca2+ regulation mechanisms during application of lactate were studied using rapid cooling contractures (RCCs) and fast application of caffeine. Cytoplasmic Ca2+ was monitored using the fluorescent indicator indo-1. 3. After application of 20 mM lactate for 5 min, the diastolic level of Ca2+ was increased. The change in cytoplasmic Ca2+ elicited by stimulation (Ca2+ transient) was also changed. With lactate, the amplitude of the Ca2+ transient was smaller, and its time course was slower compared with control. 4. The recovery of cytoplasmic Ca2+ during rewarming after rapid cooling in lactate was slower than under control conditions. When the rewarming was performed either in Na(+)- and Ca(2+)-free solution or in the presence of 10 mM caffeine, the rate of recovery of cytoplasmic Ca2+ in lactate was slower than under control conditions, suggesting that the activity of both SR Ca2+ uptake and Na(+)-Ca2+ exchange is affected by lactate. 5. Cytoplasmic Ca2+ recovery during application of 10 mM caffeine in lactate was slower than in the control. The rate of recovery of the caffeine-induced transient inward current was also slower supporting the hypothesis of a slower Ca2+ extrusion brought about by Na(+)-Ca2+ exchange. 6. The relative contribution of the Ca2+ extrusion mechanisms in the presence of lactate was investigated using paired RCCs. In lactate, a second RCC (RCC2) induced immediately after recovery from the first (RCC1) was greatly reduced compared with the control. RCC2/RCC1 x 100 in lactate was 39% and RCC2/RCC1 x 100 in control conditions was 60%, suggesting that the net sarcoplasmic reticulum Ca2+ uptake is smaller in the presence of lactate. 7. When Na(+)-free Ca2+ solution was used during the paired RCCs and rewarming, RCC2/RCC1 x 100 was increased to 96 and 95% in lactate and control conditions, respectively, implying that Ca2+ efflux from the cell can be maintained by the Na(+)-Ca2+ exchanger and that other Ca2+ removal mechanisms (mitochondria and sarcolemmal Ca(2+)-ATPase) remain largely unchanged in the presence of lactate.