The M 2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[ 3H] cis-methyldioxolane ([ 3H]CD), which has been used to label a high affinity population of M 2 receptors. A single population of sites ( K D 2.74 n m; B max of 82 fmol/mg protein) was detected and [ 3H]CD binding was sensitive to the M 2 antagonist himbacine but much less so to pirenzepine, the M 1 antagonist. These cardiac receptors had different sensitivities to NiCl 2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [ 3H]CD and considered to be of the M 2 subtype. Up to 70% of the [ 3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [ 3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K 0.5 values of 0.8, 2, 20, and 50 n m, respectively. The apparent competitive nature of inhibition of [ 3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting <50% of 5 n m [ 3H]CD binding by 1 μM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [ 3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M 2 receptors to certain OPs.