[3H]-inositol phosphate formation and the inhibition of isoproterenol-induced [3H]-cyclic adenosine monophosphate (AMP) formation in response to the muscarinic agonist carbachol were studied in cultured human airway smooth muscle cells. Stimulation with carbachol produced concentration-dependent inhibition of isoproterenol (1 microM)-induced cyclic AMP formation (EC50, 0.15 microM; maximal inhibition, 60%). This response was itself reversed by pertussis toxin (100 ng/ml) and was competitively inhibited by the muscarinic antagonists pirenzepine (pA2, 6.5), methoctramine (pA2, 8.0), 4-diphenylacetoxy-N-methyl-piperidine (pA2, 8.0), and parafluorohexahydrosiladifenidol (pA2, 6.5), indicating that the M2 receptor subtype was mediating this response. In addition, carbachol also induced [3H]-inositol phosphate formation in these cells (EC50, 11 microM; 2.1-fold stimulation over basal), although the response observed was markedly down-regulated compared with the response seen in noncultured airway smooth muscle preparations. Growth arrest of cells failed to increase the magnitude of the inositol phosphate response to carbachol. These results demonstrate that cultured human airway smooth muscle cells express functionally coupled M2 receptors and probably also low levels of coupled M3 receptors.