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We compared the binding properties of selective muscarinic antagonists with their potencies for antagonizing muscarinic responses in Chinese hamster ovary (CHO) cells expressing M2 and M3 muscarinic receptors in combination and in isolation. When measured by the competitive displacement of [3H]N-methylscopolamine binding to CHO cells expressing both M2 and M3 muscarinic receptors (CHO M2+M3 cells), the competition curves of the subtype-selective muscarinic antagonists were consistent with a two-site model. One site exhibited binding properties identical to those of CHO M2 cells, whereas the other site exhibited properties like those of CHO M3 cells. Oxotremorine-M, a muscarinic agonist, elicited a robust, pertussis toxin-insensitive stimulation of phosphoinositide hydrolysis in both CHO M3 and CHO M2+M3 cells, but not in CHO M2 cells. The pharmacological antagonism of the phosphoinositide response exhibited similar properties in both CHO M3 and CHO M2+M3 cells. Oxotremorine-M elicited a pertussis toxin-sensitive, robust inhibition of forskolin-stimulated cyclic AMP (cAMP) accumulation in both CHO M2 and CHO M2+M3 cells and a less robust inhibition in CHO M3 cells. At higher concentrations, oxotremorine-M elicited an increase in cAMP accumulation over the maximal inhibition noted at lower concentrations in both CHO M3 and CHO M2+M3 cells. Following pertussis toxin treatment, only the stimulatory phase of the cAMP response to oxotremorine-M was observed in CHO M2, CHO M3, and CHO M2+M3 cells. The pharmacological antagonism of the cAMP response in CHO M2+M3 cells resembled that expected for a response mediated independently by both M2 and M3 receptors. [Copyright &y& Elsevier]