Abstract: OBJECTIVE To explore and establish simple and reliable method of isolating single calcium-tolerant ventricular myocytes of KM mouse for patch clamping and recording of action potential and the currents of L-type calcium channels. METHODS The three-step enzymatic dissociation method was used to isolate myocytes from ventricular tissue by the Langendorff apparatus. Hearts were perfused retrogradely with Ca²⁺ free Tyrode’s solution initially, and then with Ca2+ free Tyrode’s solution contained 0.1 mg·mL^-1 collagenase Ⅱ, 0.01 mg·mL^-1 trypsin and 0.2 mg·mL^-1 bovine serum albumin. During the perfusions, 20 mL 20 mmol·L^-1 of CaCl₂ was added to the digestive juice every 5 minutes. The terminal of digestion was judged by observing the existence of single myocyte in the efflux solution. KB solution contained 1 mg·mL^-1 bovine serum albumin was ultimately used. The action potential and the currents of L-type calcium channels were recorded by patch clamp in the entire cell mode. RESULTS The 80%-90% cells obtained were rod-shaped myocytes. After the recalcification, 60% cells stayed still, and the action potentials and L-type calcium channel currents could be successfully recorded. CONCLUSION The method is economical and effective, the myocytes obstaied in this way are suitable for the recording of patch clamp technique.