Acetylcholinesterase (AChE), the enzyme that degrades acetylcholine, exists as multiple molecular forms that differ in their quaternary structure and mode of attachment to the cell surface. The distribution of the individual molecular forms of AChE in various cardiac regions with distinct anatomical characteristics was investigated. The results confirmed those of others by showing that the total pool of cardiac AChE had a nonuniform distribution in heart that paralleled the distribution of choline acetyltransferase. The rank order of this distribution was right a trial appendage >interatrial septum > left atrial appendage=right ventricle=interventricular septum > left ventricle. Velocity sedimentation in sucrose gradients of extracts from selected cardiac areas showed that four molecular forms were present in all areas but that the proportions of these forms differed as a function of area. The right and left ventricular walls, the apical portion of the Interventricular septum, and the left atrial appendage contained G, and G4(globular) AChE in near-equal proportions, but in the basal portion of interventricular septum, the contribution of G4AChE was greater than that of G1AChE. The right atrial appendage and the interatrial septum had the largest amount of activity attributable to G4AChE and the lowest amount attributable to G1AChE. In all cardiac regions, A12(asymmetric) AChE comprised 8–10% of the total AChE pool. The results of this research show that the composition of the globular AChE pool varies in heart as a function of region and that cardiac regions that have the highest AChE activity and that contain parasympathetic ganglia and nodal areas have different pools of globular AChE molecules than other areas. In addition, it appears that there is a uniform proportion of A12AChE in all areas of rat heart.