Objective To improve the method of isolation of cardiomyocytes in guinea pigs and rats for patch clamp research.Method Cardiomyocytes were prepared by improved flow-maintained Langendorff perfusion method.The currents of potassium channels were recorded by whole-cell patch clamp.Results There were 50% calcium-tolerant cardiomyocytes after exposed to normal calcium concentration solution.A current of potassium channel could be recorded.Conclusion Cardiomyocytes with electrophysiological action can be obtained by the observation of perfusion pressure variation though flow-maintained Langendorff perfusion method.