Acetylcholine (ACh) is well known as a neurotransmitter in both the central and peripheral nervous systems in mammalian species. Both muscarinic and nicotinic ACh receptors have been identified in lymphocytes isolated from thymus, lymph node, spleen, and peripheral blood, and their stimulation by muscarinic and nicotinic agonists elicits a variety of functional and biochemical effects. On the basis of these findings, it has been postulated that the parasympathetic nervous system may play a role in immune-neurohumoral crosstalk. However, ACh present in the blood of several species has been localized to lymphocytes from various origins using radioimmunoassay. Moreover, using Northern blots or reverse transcription-polymerase chain reaction, expression of choline acetyltransferase, an ACh synthesizing enzyme, has been identified in human blood mononuclear leukocytes, human leukemic T-cell lines, and rat lymphocytes. Stimulation of T-lymphocytes with phytohemagglutinin activates the lymphoid cholinergic system, as evidenced by increased synthesis and release of ACh, increased acetylcholinesterase activity, and the increased expression of mRNA encoding choline acetyltransferase and ACh receptors. The observation that muscarinic receptor stimulation by ACh or agonists increases in [Ca(2)+](i) and up-regulates c-fos expression strongly argues that ACh synthesized and released from T-lymphocytes acts as an autocrine and/or paracrine factor regulating immune function. In summary, these data present a compelling picture in which immune function is not only regulated by the cytokine system, but is also under the control of an independent, lymphoid cholinergic system.