Choline acetyltransferase (CAT) catalyzes the biosynthesis of acetylcholine according to the chemical equation: Acetyl-CoA + Choline in equilibrium Acetylcholine + CoA. To demonstrate the neuronal relationship of this enzyme, CAT activity was measured in hearts to which the extrinsic innervation was completely interrupted by extirpation of the heart from a donor animal and transplantation in a heterotopic position into the recipient's abdominal cavity. After the animals were killed at 8 days, enzyme activity in the transplanted hearts was measured and compared with that of the recipient. Choline acetyltransferase activity decreased 98% in the right atrium of the transplanted denervated hearts, 94% in the SA node, 87% in the left atrium, 80% in the right ventricle, and about 50% in the interventricular septum and left ventricle. These data show that sectioning the extrinsic parasympathetic innervation to the heart results in variable decreases in CAT activity, a finding which confirms that CAT activity is related to the extrinsic cholinergic innervation. The extrinsic (preganglionic) component of the parasympathetic system is nonuniform and supplies predominantly the specialized regions of the heart with conduction tissue. The residual enzyme activity is related to intrinsic (postganglionic) parasympathetic neurons. The intrinsic component is uniformly distributed to both specialized and contractile regions of the heart.