Src-family tyrosine kinases are signaling intermediates in a diverse array of cellular events including cell differentiation, motility, proliferation, and survival. In non-airway smooth muscle cells muscarinic receptors directly interact with src-family tyrosine kinases. As little is known about the expression and signaling of these Src family tyrosine kinases in human airway smooth muscle cells, we determined the expression of Src-family members and characterized the muscarinic receptor-mediated activation of Lyn kinase in these cells. RT-PCR revealed mRNA transcripts for FYN, c-SRC, YES, FRK and LYN. Fyn, c-Src, Yes and Lyn were identified in cultured airway smooth muscle cells by immunoblot analysis. In both native and human cultured airway smooth muscle cells transduced with wild type human Lyn kinase, carbachol increased Lyn kinase activity. Pertussis toxin pretreatment failed to block carbachol activation of Lyn kinase but did attenuate the carbachol-induced increase in ERK/MAPK phosphorylation and carbachol failed to increase total inositol phosphate synthesis in these cells. The present study shows that Lyn kinase is expressed in human cultured airway smooth muscle cells at both the mRNA and protein level and that carbachol, an M2 muscarinic receptor agonist in these cells, activates Lyn kinase by a pertussis toxin-insensitive signaling pathway.