We studied the developmental sequence of transmitter synthesis and neurotransmission by parasympathetic neurons in rat heart. Measurements of atrial and ventricular choline acetyltrans- ferase activity were used as indicators of acetylcholine synthesis, and negative chronotropic effects of neuronal stimulation were used to demonstrate neuroeffector transmission. Activity of the mitochon- dria! enzyme, carnitine acetyltransferase, was measured separately to avoid spuriously high apparent choline acetyltransferase activity. Activity of the acetylcholine synthetic enzyme, choline acetyltrans- ferase, first was detected in rat hearts on embryonic day 19, and the levels of the enzyme activity increased during the first and third weeks after birth, with levels in the atria always appearing higher than in the ventricles. In contrast, carnitine acetyltransferase activity was higher in the ventricles than in the atria, and the activity levels increased continuously from embryonic day 18 to postnatal day 21, the last day measured. Neuroeffector transmission first appeared in the rat heart on embryonic day 21, the day of birth, and was blocked by atropine. Newborn and adult hearts had similar acetylcholine sensitivity (ED50of 100 DM). Embryonic rat hearts showed slightly higher acetylcholine sensitivity. The avian class was used for comparison, and choline acetyltransferase activity first appeared in embryonic chicks on day 11. The pattern of neurotransmitter synthesis and function was identical in the avian and mammalian species, although the mammal is less mature at birth. Our studies have established the time course for development of the synthetic enzyme for acetylcholine and subsequent neurotransmis- sion in the rat heart. Circ Res 46: 690-695, 1980