We previously showed that blood acetylcholine(ACh)originates mainly from T−lymphocytes, and that stimulation of muscarinic ACh receptors(mAChRs)induces Ca²⁺ oscillations and up−regulates c−fos gene expression in both T− and B−lymphocytes.In the present study, we investigated which mAChR subtypes are involved in Ca²⁺ signaling and c−fos gene expression in human T−(CEM)and B−(Daudi)cells.Stimulation of mAChRs with 100μM oxotremorine−M, an M₁/M₃ agonist, increased levels of intracellular free Ca²⁺([Ca²⁺]_i)and c−fos mRNA expression in both cell lines.4−DAMP, an M₃ antagonist, more effectively blocked the oxotremorine−M−induced increase in [Ca²⁺]_i than pirenzepine and telenzepine, M₁−receptor antagonists;AF−DX 116, an M₂ antagonist;hexahydrosiladifenidol, a weak M₃ antagonist;or hexamethonium and d−tubocurarine, nicotinic receptor antagonists.McN−A−343(100μM), a partial M₁−receptor agonist, had no apparent effect on [Ca²⁺]_i in either cell line.The oxotremorine−M−induced up−regulation of c−fos transcription was inhibited by 4−DAMP, but not by pirenzepine or AF−DX 116.Our findings thus suggest that ACh released from T−lymphocytes acts as an autocrine/paracrine factor, transmitting a Ca²⁺−dependent signal to the nuclei of T− and B−lymphocytes via M₃ receptors.