The present study aimed to determine the effect Of two M-2/M-4-selective muscarinic receptor antagonists on blocking the hydrolysis of carbachol (CCh) stimulated phospho-inositide (PI) breakdown in order to address the possibility that a muscarinic receptor other than the M-3 receptor is involved in PI hydrolysis in this tissue. Gallbladder tissue slices labeled with myo-[2-H-3] inositol were incubated with increasing concentrations of antagonists and agonist. After the reactions were terminated by the addition of chloroform/methanol, labeled inositol phosphates were separated using anion exchange chromatography. Muscarinic M-2 antagonists methoctramine and gallamine both inhibited carbachol-induced PI breakdown at high concentrations, with 109 IC50 values of -5.145 and -6.049, respectively. Gallamine at 10(-5) M concentration failed to displace the dose-response curve for carbachol-induced accumulation of inositol triphosphate (IP3). Our data suggest that M-3 receptors play a major role in stimulation of PI hydrolysis in the guinea-pig gallbladder. (c) 2005 Elsevier Inc. All rights reserved.