OBJECTIVE In order to get high-yield and high-quality cardiomyocytes from adult rat. We needed to clarify the optimal perfusion pressure in a constant flow condition and stopping digestion pressure. METHODS The rat was injected with heparin (1 000 IU/kg, i. p.) 20 minutes prior to the experimental protocol. The heart was excised and the aorta was cannulated rapidly. The cannulated heart was mounted on a Langendorff perfusion apparatus with constant flow and perfusion pressure was monitored. The initial perfusion pressure was maintained at 15 kPa or 10 kPa by regulating the flow rate. The heart was digested by 0.08 % collagenase I at 37 degrees C and the enzymatic digestion was terminated immediately when the perfusion pressure was lowered to 10 kPa, 7.5 kPa or 5 kPa. RESULTS While the initial perfusion pressure was 15 kPa (n = 4), enzymatic digestion induced great extent increment in perfusion pressure (maximal pressure > 25 kPa). The left ventricle was enlarged significantly and the contractility of myocytes was decreased. While the initial perfusion pressure was kept at 10 kPa, the digestion only resulted in less extent increment in perfusion pressure (maximal pressure < 18.75 kPa). The left ventricle was enlarged slightly. In the same initial perfusion pressure, if the digestion was stopped at 10 kPa (n = 3), or 5 kPa (n = 4), myocytes appeared over much or insufficiency digestion, respectively. The viability of freshly isolated cardiomyocytes was less than 10%. The most of myocytes died after restoration of extracellular Ca2+ concentration. If the digestion was stopped at 7.5 kPa (n = 15). The viability of cardiomyocytes was 82.6% +/- 4.8% in fresh isolation, 30.4% +/- 4.5% after restoration of normal extracellular Ca2+ concentration, or 24.8% +/- 5.4% after 4 h standing. The most of rod-shaped cardiomyocytes were quiescent and had visible cross striations, sharp edges, and normal contractility. CONCLUSIONS When the initial perfusion pressure is kept at 10 kPa (low pressure) and the enzymatic digestion is stopped at 7.5 kPa, high yield and high quality cardiomyocytes can be obtained.