Contraction of the guinea pig taenia caeci is mediated by muscarinic M3 receptors; however, they comprise only 30% of the muscarinic receptors present. This study investigated the role of the predominant M2 receptor population in contractions and possible second messengers involved after M3 receptors were selectively alkylated by 4-DAMP mustard [N-(2-chloroethyl)-4-piperidinyldiphenylacetate] (60 nM) in the presence of otenzepad (AF-DX 116; 1 microM). Concentration-response curves to oxotremorine-M (oxo-M) in the presence of histamine and isoprenaline were performed in the presence of otenzepad (1 and 3 microM), resulting in a mean apparent pK(B) of 6.49, indicative of an M2 response. As the taenia has intrinsic tone, precontraction with histamine was not necessary and, therefore, in some experiments only isoprenaline was included. In these studies, an M3 response to oxo-M was observed, as the mean apparent pK(B) for otenzepad was 5.89. To investigate protein kinase C (PKC) involvement in the M2 response following M3 inactivation, the inhibitor chelerythrine (1 microM) was included with histamine and isoprenaline in the absence and presence of otenzepad. The oxo-M concentration-response curve was shifted by otenzepad with an apparent pK(B) value of 6.05, a value significantly different from that seen in the absence of chelerythrine (P < 0.05). These results suggest that activation of PKC by a spasmogen such as histamine is necessary to see an M2 response following M3 receptor inactivation.