The effects of growth factors on cell growth and muscarinic receptor (mAChR) expression of canine tracheal smooth muscle cells (TSMCs) were observed under serum-free medium supplemented with 0.1% BSA. In the presence of 0.1% BSA, TSMCs withdraw from cell cycle as compared with 10% FBS and allow to determine the effects of growth factors on mAChR expression. The individual components of growth factors (IGF-I, insulin, and aFGF) at the concentration used are not sufficient to stimulate growth of TSMCs in the primary culture with 0.1% BSA. IGF-I (10 ng/ml) and insulin (1 microgram/ml), alone or in combination, could stimulate the expression of mAChRs of cultured TSMCs. Heparin could inhibit these stimulatory effects of mAChR expression. The stimulatory effects of IGF-I and insulin on mAChR expression were mediated through their own receptors since these effects were reversed by pretreatment of TSMCs with antibodies of the respective growth factor receptors. The pharmacological response of functional mAChRs, determined as accumulation of inositol phosphates induced by carbachol, is greater in the medium containing IGF-I and insulin than that cultured in 0.1% BSA. These results firmly establish that IGF-I and insulin could stimulate the expression of mAChRs in TSMCs under serum-free culture condition.