The pharmacological characteristics of muscarinic receptor (mAChR) subtypes in canine left ventricular membranes (LVM) were determined using [3H] quinuclidinyl benzilate ([3H]QNB) and [3H]N-methyl scopolamine ([3H]NMS) as ligands. Binding of [3H]QNB and [3H]NMS was saturable with respect to the radioligand concentrations. Analysis of binding isotherms by Scatchard plot showed that [3H]QNB and [3H]NMS bound to an apparently homogeneous population of mAChRs in LVM, with KD values of 390 +/- 100 and 285 +/- 34 pM and Bmax values of 240 +/- 20 and 133 +/- 9 fmol/mg protein, (n = 6), respectively. The Hill coefficients for [3H]QNB and [3H]NMS binding were 0.95 +/- 0.02 and 0.99 +/- 0.01, respectively. Based on the competitive inhibition of [3H]ligand binding, atropine and NMS as well as the selective M1 antagonist PZ revealed no selectivity for these mAChRs. PZ competed with [3H]QNB or [3H]NMS for a single binding site with a Ki value of 0.23 +/- 0.03 microM and 0.62 +/- 0.10 microM, (n = 6), respectively, which is close to the values of M2 or M3 receptors. The data indicate that the M1 receptor subtype did not exist in canine LVM. Competition of [3H]ligand binding with selective M2 antagonists, AF-DX 116 and methoctramine and the selective M3 antagonists, 4-DAMP and hexahydrosiladifenidol, gave a best fit for a two-binding site model. The inhibition of carbachol-mediated phosphoinositide hydrolysis by PZ, AF-DX 116 and 4-DAMP, generated an affinity profile for this response also dissimilar to that described for the classical cardiac M2 response. Although no other muscarinic receptor mRNA has been detected in this tissue, these data suggest the presence of a second population of muscarinic sites, which may signify an M2 receptor diversity.