We have studied the role of M(2) and M(3) muscarinic receptors in acetylcholine-mediated desensitization of the contractile response to histamine in the guinea pig ileum. Treatment of the isolated ileum with acetylcholine (30 microM) for 20 min caused a marked desensitization of the contractile response to histamine. When measured 5 min after washout of acetylcholine, the EC(50) value of histamine increased 5.8-fold compared with that estimated before acetylcholine treatment, whereas the maximal response was unaffected. This shift in the EC(50) value of histamine was maximal at the earliest time measured after acetylcholine treatment (5 min), and normal sensitivity recovered in approximately 20 min. Acetylcholine-induced desensitization was prevented by uncoupling of M(2) receptors from G(i) with pertussis toxin or by selective inactivation of M(3) receptors with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard). The shifts in the EC(50) values of histamine measured 5 min after acetylcholine treatment were only 2.0- and 1.8-fold in pertussis toxin- and 4-DAMP mustard-treated ilea, respectively. Both pertussis toxin- and 4-DAMP mustard-treatment had little or no effect on histamine-induced contractions in control ileum. Measurement of histamine-stimulated inositol phosphate accumulation in the longitudinal muscle of the ileum showed little or no inhibitory effect of prior exposure to acetylcholine, indicating that the majority of the heterologous desensitization occurs downstream from phospholipase Cbeta activation. Collectively, our results suggest that activation of both M(2) and M(3) receptors is required for heterologous desensitization of histamine-mediated contractions in the guinea pig ileum.