The biochemical linking event between the activation of muscarinic receptors and the inhibition of adenylate cyclase was studied with rat heart ventrical membranes. The muscarinic M2 selective antagonists were more potent than the M1 selective antagonists in the displacement of non-selective labeled 3H-QNB binding to the membranes. This was also true for the M2 selective agonists, with respect to the M1 selective agonists, in the reaction medium without Gpp(NH)p. With the same preparation, the muscarinic M2 selective agents were also more potent than the M1 selective agents in the inhibition of adenylate cyclase activity. The order of potencies of these agents in the displacement of 3H-QNB binding correlated well with their order of potencies in inhibiting adenylate cyclase activity. Gpp(NH)p reduced the binding affinities of M2 selective agonists (i.e. carbachol and oxotremorine), while it did not affect the binding affinities of non-selective agonist pilocarpine, M1 selective agonist McN-A-343 and antagonists (i.e. pirenzepine, trihexyphenidyl, AF-DX-116 and methoctramine). These results suggest that in the rat heart, the inhibition of adenylate cyclase by muscarinic agonists is through activation of the M2 subtype receptor and G-protein is likely to be involved in the coupling.