Abstract: Objective To investigate the general pattern of cholinergic nerve distribution and M2 receptors in adult rat heart. Methods Karnovsky-Roots histochemical staining combining point counting method and immunochemical SABC method with image analysis were used to identify the cholinergic nerves and M2 receptors, respectively, in adult rat heart. Results Positive staining of cholinergic nerves and M2 receptors was found in all regions of the rat heart, and the point count of cholinergic nerves in the atria was 4.6 times as much as that in ventricles, and the area of immunoreactive substance for M2 receptors two-fold higher in the atria than in the ventricles. The point counts of the cholinergic nerves in the medial-layer myocardium were fewer than that in subepicardial and endocardial tissues of the left ventricular free wall. However, M2 receptors were comparable among the 3 layers of the left free ventricular wall. Conclusion Cholinergic nerves and M2 receptors are located in both rat atria and ventricles, but their density is much higher in the atria than in the ventricles. Transmural heterogeneity characterizes cholinergic nerve innervation in the left ventricular free wall without significant differences in M2 receptor density.