In the pulmonary vasculature of man, pig and guinea-pig, acetylcholine (ACh) exerts a relaxant effect by interacting with muscarinic receptors located on endothelial cells. The present experiments were designed to detect the endogenous source of ACh in the pulmonary vasculature. For this purpose, we investigated whether pulmonary artery endothelial cells contain elements of the "cholinergic gene locus", the ACh synthesising enzyme choline acetyltransferase (ChAT) and the vesicular ACh transporter (VAChT). ChAT mRNA was detected by means of reverse transcription polymerase chain reaction (RT-PCR) in endothelial cells of porcine pulmonary arteries freshly isolated and after 7 days in culture. ChAT protein was demonstrated in endothelial cells in vitro and in situ. ChAT immunoreactivity was present in endothelial cells freshly isolated and after 2, 4, 7, and 9 days in culture. Tissue sections from extra- and intraparenchymal pulmonary arteries of man, pig and guinea-pig expressed a mosaic pattern of ChAT-positive and -negative endothelial cells. VAChT mRNA was detected by RT-PCR in rat pulmonary artery and in endothelial cells isolated from human and porcine pulmonary trunk. The detection of VAChT and ChAT mRNA and the demonstration of ChAT protein in vitro and in situ suggest that the endothelium is an endogenous source of ACh in the pulmonary vasculature.