For isolation of neonatal rat cardiomyocytes (NRCM) the ventricular muscles of neonatal rats were treated with different digestive solutions: 0.06% trypsin (method I), 0.08% collagenase II (method II), 0.06% trypsin and 0.08% collagenase II for stepwise digestion (methods III and IV). After enzymatic dissociation of the tissue, the complete medium was added to stop this process. The cells suspensions obtained by methods I-III were collected and centrifuged. In contrast, the novel and improved method IV did not use centrifugation. Instead, various methods of adhesion were employed to separate non-myocardial cells. The isolation methods were compared by the quantity, survival rate, morphology, spontaneous pulsation rate, purity, and vitality of NRCM. These assessments showed that isolation method IV is a simple, efficient, and convenient way to obtain NRCM for culturing.