The reperfusion of hearts with Ca2+-containing buffers after relatively short periods of Ca2+-free perfusion results in tissue damage, Ca2+ overload, loss of mechanical function and displacement of intracellular components into the extracellular phase. Using isolated, spontaneously beating Langendorff perfused rat hearts we have investigated whether Mn2+ and Co2+ exert a dose dependent inhibitory effect on this gain in Ca2+ and loss of intracellular constituents (assayed as myoglobin). We have also determined if the timing of the addition of Co2+ or Mn2+ is critical, and whether their protective effect is Ca2+-sensitive. When added only at Ca2+ repletion neither Co2+ nor Mn2+ provided protection. When present during the entire period of Ca2+ depletion and repletion, Mn2+ (005 to 4.0 mM) and Co2+ (0.1 to 4.0 mM) exerted a dose-dependent protective effect, indicted by a reduced and delayed onset of myoglobin release, a reduced gain in Ca2+, and an improved recovery of mechanical function. This protection was proportionally diminished if the cations were added late during the 10 min period of Ca2+ depletion and further reduced if after their late addition, Mn2+-free or Co2+-free Ca2+ repletion buffer was used. Mn2+ was more effective than Co2+, and decreasing the Ca2+ content of the perfusion buffer from 2.5 to 1.3 mmol.1-1 shifted the dose-response curve for this protection to the left. These results are discussed in terms of the possible mechanisms involved in the dose and time dependent protective effect of Mn2+ and Co2+.