The presence of M-1-M-4 receptors in guinea-pig gallbladder smooth muscle cells has been reported recently. The majority of these receptors are said to be of M-2 subtype. However, there are controversial reports about the functional muscarinic receptors that mediate contraction in this tissue. Similar to gallbladder, it was claimed that M-4 receptors mediate guinea-pig uterine contractions, but these receptors have appeared to be of M-2 subtypes later. Therefore, the antagonistic affinities of three M-2-selective muscarinic antagonists were determined in contraction and radioligand binding experiments in guinea-pig gallbladder in the present study. The antagonistic affinity Values (pK(i)) of gallamine, tripitramine and imperialine were as follows, respectively: 6.28+/-0.15, 8.65+/-0.10 and 6.55+/-0.07 against 0.250 nM [H-3]QNB binding. All three antagonists displaced the concentration-response curves to carbachol to the right in parallel without affecting the maximum responses. The pA(2) values obtained from constrained Schild plots (-log K-B) were 4.14+/-0.18 for gallamine, 6.79+/-0.09 for tripitramine, and 7.02+/-0.09 for imperialine. The antagonistic affinity values of gallamine, tripitramine and imperialine for M-2 receptors are reported to be 6.3, 9.6, 7.7, respectively. The pA(2) values obtained in this study clearly indicate that the primary muscarinic receptors involved in carbachol-induced guinea-pig gallbladder contraction are not of M-2 subtype. The poor correlation between the antagonistic affinity values of these antagonists obtained at radioligand binding (pK(i)) and contraction (pA(2)) experiments also support the conclusion that the majority of muscarinic receptors which have been reported to be of M-2 do not mediate the contractile responses. (C) 1999 Academic Press.