In hearts, intracellular acidosis disturbs contractile performance by decreasing myofibrillar Ca²⁺ response, but contraction recovers at prolonged acidosis. We examined the mechanism and physiological implication of the contractile recovery during acidosis in rat ventricular myocytes. During the initial 4 min of acidosis, the twitch cell shortening decreased from 2.3 ± 0.3% of diastolic length to 0.2 ± 0.1% (means ± SE, P < 0.05, n = 14), but in nine of these cells, contractile function spontaneously recovered to 1.5 ± 0.3% at 10 min (P < 0.05 vs. that at 4 min). During the depression phase, both the diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ transient (CaT) amplitude increased, and the twitch [Ca²⁺]_i decline prolonged significantly (P < 0.05). In the cells that recovered, a further increase in CaT amplitude and a reacceleration of twitch [Ca²⁺]_i decline were observed. The increase in diastolic [Ca²⁺]_i was less extensive than the increase in the cells that did not recover (n = 5). Blockade of sarcoplasmic reticulum (SR) function by ryanodine (10 µM) and thapsigargin (1 µM) or a selective inhibitor of Ca²⁺-calmodulin kinase II, 2-[N- (2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methyl benzylamine (1 µM) completely abolished the reacceleration of twitch [Ca²⁺]_i decline and almost eliminated the contractile recovery. We concluded that during prolonged acidosis, Ca²⁺-calmodulin kinase II-dependent reactivation of SR Ca²⁺ uptake could increase SR Ca²⁺ content and CaT amplitude. This recovery can compensate for the decreased myofibrillar Ca²⁺ response, but may also cause Ca²⁺ overload after returning to physiological pH_i.