The roles of the sarcoplasmic reticulum (SR) Ca(2+)‐ATPase and Na(+)‐Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo‐1‐loaded cells. Na(+)‐Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre‐perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady‐state twitches was 2‐fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half‐time for relaxation of caffeine contractures 15‐ and 5‐fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)‐Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)‐ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9‐fold) than in rabbit myocytes (2‐fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG‐treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)‐ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)‐Ca2+ exchange is about 2‐ to 3‐fold faster in rabbit than in rat, whereas the SR Ca(2+)‐ATPase is 2‐ to 3‐fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)