Previous reports have not suggested a critical answer for the question, if the efficacy of crystalloid cardioplegic (St' Thomas Hospital) solution against immature myocardium was the same as that against mature myocardium, or not. The author in this study, therefore, investigated 72 isolated puppy (2-4 weeks old) hearts dividing them at random into three groups according to the different doses to be administered, namely, Group A: single-dose of 10 ml/kg of cardioplegia; Group B: multiple-dose of cardioplegia with the initial dose of 10 ml/kg followed by 5 ml/kg every 20 minutes; and Group C: no use of cardioplegia. They were subjected to 120 minutes of ischemic arrest at 15 degrees C and subsequent 30 minutes of reperfusion. Ultrastructural changes of the myocardium were studied systematically and, in particular, semiquantification was carried out on mitochondria. Gravimetric and high energy phosphate (HEP) content of myocardium, and recovery of left ventricular end-systolic pressure volume ratio (Emax), spontaneous defibrillation ratio were also studied. Electron microscopic study showed that ischemic myocardial change with global ischemia was severer and more frequent in Group C as compared to Groups A and B, which coincided with less HEP content in Group C. Intracellular rarefaction following global ischemia was less frequent in Group A than in Group B or C, which corresponded to the result of gravimetric myocardial water content study. Spontaneous defibrillation in Group C was evidently prolonged, although there was no significant difference in the left ventricular function among the groups. The author should recommend single-dose administration of cardioplegia with topical cooling against immature myocardium, because this cardioplegia was effective in either single- or multiple-dose, and intracellular myocardial edema was lesser in the case of single-dose than in multiple-dose.