Single heart cells of mouse models provide powerful tools for heart research. However, their isolation is not easy, and it imposes a significant bottleneck on their use in cellular studies of the heart. Aiming to overcome this problem, this report introduces a novel technique that reproducibly isolates healthy heart cells from mouse models. Using simple devices that ensure easy handling and the rapid aortic cannulation of a small mouse heart, cell isolation was done under physiological conditions without using the “KB” medium or 2, 3-butanedione monoxime (BDM). The isolated cells consistently had a healthy appearance and a high viability of 75±5%(mean±SD) in Tyrode solution containing 1.8 mM Ca2+. After 8 h of storage at 37 C, they still had a viability of 45±12%. The cells showed normal contraction properties when field-stimulated, and they generated normal action potentials and membrane currents under the whole-cell clamp condition. The β-adrenergic signal transduction of the cells was also normal when it was examined with the isoproterenol enhancement of the L-type Ca2+ current.