The purpose of this study was to evaluate the functional and biochemical effects of cardioplegic solutions on immature cardiac myocytes incubated under hypothermic conditions. In addition, the effects of insulin as an additive were evaluated in each solution. Cardiac myocytes were isolated from neonatal rat ventricles and cultured for four days; 12.5 x 105 myocytes/flask were then incubated at 4 °C for three, six and 12 hours in three types of cardioplegic solutions—glucose-potassium solution (glucose: 50 gm/l, NaHCO3: 20 mEq, KCl: 20 mEq), lactated Ringer's solution (KCl: 20 mEq) and St. Thomas' Hospital solution. After each hypothermic incubation, enzymes were measured in the incubation solutions. The myocytes were then cultured for an additional 24 hours at 37 °C to evaluate the recovery of the myocyte beating rate. In the Ringer's group, the recovery ratio of the myocyte beating rate was complete at three hours, then decreased to 48.8 percent of control (beating rate prior to hypothermic incubation) at 12 hours. The glucose-potassium and St. Thomas' groups had significantly lower recovery ratios than the Ringer's group, beginning at three hours (63.4, 72.9, 95.6 percent, respectively). Release of enzymes (CPK and LDH) in the Ringer's group gradually increased and at 12 hours was 29.0 mIU/flask and 260.0 mIU/flask, respectively. The St. Thomas' group, in contrast, had significantly increased values for CPK at 12 hours to 116.0 mIU/flask, and the greatest increases of both enzymes were observed in the glucose-potassium group at 12 hours (CPK: 115.5, LDH: 1163.9). By addition of 20 IU/l insulin, marked improvements were observed in the Ringer's and glucose-potassium groups both functionally and biochemically. Thus, the lactated Ringer's solution had the least cytotoxic effects that might be suitable for a basic solution of various cardioplegic solutions during the neonatal period, and insulin may have beneficial effects on immature myocardium under hypothermic conditions.