Acetylcholine (ACh) is the major transmitter to induce contraction of the airway smooth muscle (ASM). In addition to the quantal vesicular ACh release after stimulation of parasympathetic neurons, non-quantal ACh (nqACh) release is observed in tracheal preparations after eserine treatment, which is capable to induce ASM contraction. In order to identify which cell population in the airways contributes to nqACh synthesis, expression of the choline acetyltransferase (ChAT), high affinity choline transporter-1 (CHT1), and the low-affinity choline transporter-like family (CTL1 – 5) was evaluated by RT-PCR in primary isolated vagal and dorsal root ganglia, parasympathetic neurons, tracheal epithelial cells, lymphocytes, granulocytes, and in a smooth muscle and fibroblast cell line. All evaluated cell populations expressed ChAT-, CHT1- or CTL-mRNA, respectively. ACh release was functionally investigated by using M3WT4 cells, a reporter cell line overexpressing muscarinic M3 receptors. An increase in [Ca2+]i was induced in M3WT4 cells by supernatants of eserine-treated tracheal preparations and inhibited by atropine co-application. However, supernatants of eserine-treated isolated cell populations or cells co-cultured cells with M3WT4 cells, respectively, had no effect on [Ca2+]i levels. To address the question if mast cells might contribute to nqACh release, organ bath experiments with tracheas of mast cell-deficient B6.Cg-KitW-sh/HNihrJaeBsmGlliJ mice (kindly provided by Frank Petersen, Leibniz Forschungszentrum Borstel, ARCN) were performed. ASM contraction in response to eserine in mast cell-deficient mice was unaltered in comparison to wild-type mice. In conclusion, we were able to show that many different cell populations located in the trachea express transporters and/or enzymes associated with nqACh release. The fact that we were not able identify the major source of nqACh may be due to a low sensitivity of the M3WT4 cell assay. Alternatively, the possibility that the effect of nqACh-dependent ASM contraction is the sum of low-level ACh released from different cell populations has to be discussed.