Abstract Background Chronic airway diseases, like asthma or COPD, are characterized by excessive acetylcholine release and airway remodeling. The aim of this study was to investigate the long-term effect of muscarinic agonists on the phenotype and proliferation of rabbit tracheal airway smooth muscle cells (ASMCs). Methods ASMCs were serum starved before treatment with muscarinic agonists. Cell phenotype was studied by optical microscopy and indirect immunofluorescence, using smooth muscle α-actin, desmin and SM-Myosin Heavy Chain (SM-MHC) antibodies. [N-methyl-³H]scopolamine binding studies were performed in order to assess M₃ muscarinic receptor expression on isolated cell membranes. Contractility studies were performed on isolated ASMCs treated with muscarinic agonists. Proliferation was estimated using methyl-[³H]thymidine incorporation, MTT or cell counting methods. Involvement of PI3K and MAPK signalling pathways was studied by cell incubation with the pathway inhibitors LY294002 and PD98059 respectively. Results Prolonged culture of ASMCs with acetylcholine, carbachol or FBS, reduced the expression of α-actin, desmin and SM-MHC compared to cells cultured in serum free medium. Treatment of ASMCs with muscarinic agonists for 3-15 days decreased muscarinic receptor expression and their responsiveness to muscarinic stimulation. Acetylcholine and carbachol induced DNA synthesis and increased cell number, of ASMCs that had acquired a contractile phenotype by 7 day serum starvation. This effect was mediated via a PI3K and MAPK dependent mechanism. Conclusions Prolonged exposure of rabbit ASMCs to muscarinic agonists decreases the expression of smooth muscle specific marker proteins, down-regulates muscarinic receptors and decreases ASMC contractile responsiveness. Muscarinic agonists are mitogenic, via the PI3K and MAPK signalling pathways.