The turnover of neurotransmitter is a direct measure of neuronal function, varying with the impulse activity of the nerve. It is not known if vagal stimulation increases acetylcholine release uniformly throughout the heart, or if modification of neural signals occurs between the vagal nerve trunks and postganglionic synaptic terminals. The rate constant of acetylcholine turnover was measured in conduction and contractile regions of heart by quantifying the incorporation of [3H]choline into acetylcholine after labeling of the blood choline pool in urethane-anesthetized rats during two levels of vagal activity. Choline and acetylcholine were assayed by high pressure liquid chromatography with electrochemical detection of post-column enzymic reaction product, peroxide. The specific activities of choline and acetylcholine in the tissues at sacrifice were used to calculate the fractional turnover rates in cardiac regions. Supramaximal bilateral vagal stimulation for 20 minutes decreased heart rate (P less than 0.05), while mean arterial blood pressure remained constant. The rate constants for acetylcholine turnover in right atrial regions containing the sinoatrial node, left atrial tissues, and interatrial septum doubled from control values during vagal stimulation. In contrast, the fractional rate constants of acetylcholine turnover did not change in the right and left ventricles during vagal stimulation. We interpret these results to indicate general activation of postganglionic parasympathetic fibers to the atria and selective modulation of postganglionic parasympathetic neural function to the ventricles.