In order to clarify the origin of acetylcholine (ACh) in human blood, we measured the content and synthesis activity of ACh in several human leukemic cell lines. The intracellular ACh content determined by a specific and sensitive radioimmunoassay in the human leukemic T cell lines, HSB-2, MOLT-3, and CEM, was 79.6, 36.2, and 9.5 pmol/106 cells, respectively. These values were 9-70-fold higher than those of other cell lines, including a helper T cell line, Jurkat. Stimulation of HSB-2 and MOLT-3 by phytohemagglutinin (PHA) increased both the intracellular content and release of ACh into the culture medium, but did not influence the intracellular content and release of ACh in CEM. ACh synthesis activity was found in all the T cell lines tested. Bromoacetylcholine (100 pM), a choline acetyltransferase (ChAT) inhibitor, and bromoacetyl-L-carnitine (100 pM), a carnitine acetyltransferase (CarAT) inhibitor, decreased AChsynthesizing activity in MOLT-3, and HSB-2 and CEM, by about 50% and 30%, respectively, indicating that both ChAT, and to a lesser extent CarAT, are involved in ACh synthesis in T cells. These results suggest that T lymphocytes have the potential to synthesize and release ACh, which may play a role in regulating T cell-dependent immune responses.