The purpose of the present study was to evaluate the functional and biochemical effects of serum concentrations on hypothermic preservation of cardiac myocytes. Myocytes were isolated from neonatal rat ventricles and cultured for 4 days, and then incubated for 24 hrs at 4 degrees C in media containing 0, 2, 5, 10, and 20% fetal calf serum (FCS). The myocytes were then cultured for an additional 24 hrs at 37 degrees C to evaluate the recovery of myocyte beating rate. The recovery ratio of myocyte beating rate after hypothermic incubation was 25.3 +/- 14.2 percent of control in the serum-free, 33.0% in 2% FCS, 30.7% in 5% FCS, 22.3% in 10% FCS, and 24.3% in 20% FCS groups. Serum-free, 10% and 20% FCS groups had lower recovery ratios compared to the other two groups. The release of CPK and LDH after hypothermic incubation were similar in the serum-free, 2% and 5% FCS groups, however, significant increases were observed for the 10% and 20% FCS groups. It is suggested that fetal calf serum at high concentrations (> 10%) might be injurious to immature myocardium under hypothermic preservation, and that the optimum concentration of FCS is two percent or less.