AIM:To establish and compare the methods for  culturing neonatal rat cardiac fibroblasts culture. METHODS:Neonatal rat hearts were isolated by collagenase+trypsin or trypsin digestion. The cardiomyocytes and cardiac fibroblasts were isolated by different attachment techniques. The cellular morphology, purity and reactions to the reagent were tested. RESULTS:For morphology and purity, no difference between the 2 methods was observed, though more myocytes were harvested by the method of collagenase+trypsin digestion. For the cellular responses to reagent, the cardiac fibroblasts harvested with trypsin digestion had more potent proliferative ability than those with collagenase+trypsin digestion. CONCLUSION:There is no difference of cellular morphology and purity in the cardiac fibroblasts isolated by collagenase+trypsin digestion and trypsin digestion, but the fibroblasts with trypsin digestion have more potent proliferative ability.