A liquid chromatographic assay was used to determine the choline and acetylcholine concentrations in the four chambers of rat hearts 2, 4, and 8 days after transplantation into an abdominal site. Corresponding measurements were made in the hearts of host rats. We found regional cardiac acetylcholine concentrations in controls follow the nonuniform pattern seen with choline acetyltransferase (CAT) activity, being highest in the atria (8-12 nmol/g) and lower in the ventricles (0.7-1.6 nmol/g). Following transplantation, acetylcholine levels decreased significantly only in the right ventricle after 8 days. Following a unilateral vagotomy (random, left or right), acetylcholine concentrations in the distal portion of the severed nerve decreased to half the value of the intact contralateral side by 4 days. The continued presence of acetylcholine, despite significantly reduced CAT activity in the severed nerve and transplanted heart, suggests that acetylcholine is preserved, perhaps by vesiculation in nonstimulated postganglionic terminals. The localized decrease in acetylcholine in the right ventricle after 8 days suggests that transplantation may interrupt the postganglionic fibers to this area.