Muscarinic agonists elicit contraction through M3 receptors in most isolated preparations of gastrointestinal smooth muscle, and not surprisingly, several investigators have identified M3 receptors in smooth muscle using biochemical, immunological and molecular biological methods. However, these studies have also shown that the M2 receptor outnumbers the M3 by a factor of about four in most instances. In smooth muscle, M3 receptors mediate phosphoinositide hydrolysis and Ca2+ mobilization, whereas M2 receptors mediate an inhibition of cAMP accumulation. The inhibitory effect of the M2 receptor on cAMP levels suggests an indirect role for this receptor; namely, an inhibition of the relaxant action of cAMP-stimulating agents. Such a function has been rigorously demonstrated in an experimental paradigm where gastrointestinal smooth muscle is first incubated with 4-DAMP mustard to inactivate M3 receptors during a Treatment Phase, and subsequently, the contractile activity of muscarinic agonists is characterized during a Test Phase in the presence of histamine and a relaxant agent. When present together, histamine and the relaxant agent (e.g., isoproterenol or forskolin) have no net contractile effect because their actions oppose one another. However, under these conditions, muscarinic agonists elicit a highly potent contractile response through the M2 receptor, presumably by inhibiting the relaxant action of isoproterenol or forskolin on histamine-induced contractions. This contractile response is pertussis toxin-sensitive, unlike the standard contractile response to muscarinic agonists, which is pertussis toxin-insensitive. When measured under standard conditions (i.e., in the absence of histamine and without 4-DAMP mustard-treatment), the contractile response to muscarinic agonists is moderately sensitive to pertussis toxin if isoproterenol or forskolin is present. Also, pertussis toxin-treatment enhances the relaxant action of isoproterenol in the field-stimulated guinea pig ileum. These results demonstrate that endogenous acetylcholine can activate M2 receptors to inhibit the relaxant effects of beta-adrenoceptor activation on M3 receptor-mediated contractions. An operational model for the interaction between M2 and M3 receptors shows that competitive antagonism of the interactive response resembles an M3 profile under most conditions, making it difficult to detect the contribution of the M2 receptor.