Using cultured endothelial cells prepared from bovine carotid artery, and a specific radioimmunoassay for acetylcholine (ACh), the synthesis and release of ACh by vascular endothelial cells were investigated directly. ACh content in the culture medium after 24 h of incubation in the presence of isoflurophate, a nonspecific cholinesterase inhibitor, was about 16 times higher than that in endothelial cells, indicating that ACh synthesized inside the cells was released rapidly. The presence of ACh in the culture medium was further confirmed qualitatively using high-performance liquid chromatography with an electrocapture detection. These results represent the first direct evidence that endothelial cells can synthesize and release ACh, suggesting the possibility that ACh acts not only as a neurotransmitter but also as an autacoid under certain conditions.