Polyamine levels were measured by means of high-performance liquid chromatography in Langendorff-perfused rat hearts subjected to the calcium paradox protocol. The concentrations of putrescine, spermidine and spermine did not change significantly during calcium-free perfusion but decreased when calcium was readmitted. This decrease was due to membrane disruption and release of the polyamines into the coronary effluent. The sum of released and remaining spermidine exceeded the concentration of spermidine in control hearts, but, for spermine, this sum was lower than the control level. The addition of 0.5 mM EGTA to the calcium-free solution raised the myocardial concentrations of putrescine and spermidine and enhanced the net increase of spermidine on calcium repletion. DL-alpha-Difluoromethylornithine (DFMO) inhibited these increases and lowered the putrescine level during all perfusion stages. External polyamines had a negative inotropic effect and inhibited the loss of myoglobin on calcium repletion (order of effectiveness: spermine greater than spermine greater than putrescine). Inhibition of contractions by the combined action of verapamil and ryanodine or by potassium depolarization did not prevent myoglobin loss. External polyamines had no effect on high K/low Na contractures, which were mediated mainly by Na-Ca exchange. Calcium-free perfusion in the presence of 0.5 to 1 mM EGTA improved the membrane protection by polyamines or by diamines and analogues, like ornithine, 1,3-diaminopropane, or DFMO, which, in the absence of EGTA, gave no clear protection. It is concluded that calcium depletion and repletion influences myocardiaal polyamine concentrations by (1) membrane disruption and release of polyamines into the coronary effluent, and (2) probably by a stimulation of ornithine decarboxylase activity. The changes in polyamine concentrations do not seem to have any causal role in calcium overload and cell death. Exogenous polyamines protect against membrane damage.