We studied relaxation during rapid rewarming of rabbit ventricular muscles that had been activated by rapid cooling. Rewarming from 1 degree to 30 degrees C (in less than 0.5 second) activates mechanisms that contribute to the reduction of intracellular calcium concentration and thus relaxation (e.g., sarcoplasmic reticulum [SR] calcium pump and sarcolemmal Na-Ca exchange and calcium pump). Rapid rewarming in normal Tyrode's solution induces relaxation with a half-time (t1/2) of 217 +/- 14 msec (mean +/- SEM). During cold exposure, changing the superfusate to a sodium-free, calcium-free medium with 2 mM CoCl2 (to eliminate Na-Ca exchange) slightly slows relaxation upon rewarming in the same medium (t1/2 = 279 +/- 44 msec). Addition of 10 mM caffeine (which prevents SR calcium sequestration) to normal Tyrode's solution during cold superfusion slows relaxation somewhat more (t1/2 = 376 +/- 31 msec) than sodium-free, calcium-free solution. However, if both interventions are combined (sodium-free + caffeine) during the cold exposure and rewarming, the relaxation is greatly slowed (t1/2 = 2,580 +/- 810 msec). These results suggest that either the SR calcium pump or, to a lesser extent, sarcolemmal Na-Ca exchange can produce rapid relaxation, but if both systems are blocked, relaxation is very slow. If muscles are equilibrated with 500 nM ryanodine before cooling, relaxation upon rewarming is not greatly slowed (t1/2 = 266 +/- 37 msec) even if sodium-free, calcium-free solution is applied during the cold and rewarming phases (t1/2 = 305 +/- 66 msec). This result suggests that ryanodine does not prevent the SR from accumulating calcium to induce relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)