This article examines the role of innervation in regulating expression of acetylcholinesterase (AchE), butyrylcholinesterase (BuchE), and the muscarinic acetylcholine receptor (mAchR) in avian heart. Two distinct approaches are taken. The first approach examines the relation between the onsets of parasympathetic and sympathetic innervation and the appearance of AchE and BuchE. All molecular forms of AchE and BuchE are present in early embryonic chick heart well before the onset of parasympathetic and sympathetic innervation. These molecular forms are characterized by sedimentation coefficients of 4.5S, 11S, 15S, and 19S. With further development, the amounts of AchE fall; the reductions in AchE parallel the onset of functional parasympathetic innervation. The amounts of BuchE increase progressively throughout embryonic development, independent of autonomic innervation, and in mature chick heart predominate over the much less abundant amounts of AchE. The 15S and 19S forms of AchE in heart are lost during early embryogenesis but reappear in skeletal muscle during later embryogenesis. The second approach examines the influence of vagotomy and sympathetic denervation of 8-day-old chick myocardium on expression of the molecular forms of AchE, BuchE, mAchR, and β-adrenergic receptors. The amounts of AchE and BuchE molecular forms in avian heart are not measurably influenced by bilateral vagotomy for a duration of 4 days, unilateral vagotomy for a duration of 25 days, or sympathetic denervation. A measurable upregulation is observed in muscarinic receptors (35–46%) after vagotomy but not sympathectomy and in β-adrenergic receptors (29%) after sympathectomy but not vagotomy. In all cases, results in atria and ventricles are nearly identical. The present results indicate that expression of AchE in the myocardium is unique and different from that in skeletal muscle and not directly linked with autonomic innervation.