AbstractMuscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non‐selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype‐selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD= 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains.In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype‐selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1selective antagonist 11‐((2‐((dimethylamino)‐methyl)1‐ piperidinyl)acetyl)‐5, 11‐dihydro‐6H‐pyrido(2,3‐b)‐(1,4)‐benzodiazepin‐6 one (AF‐DX 116), the M2‐selective antagonist, and 4‐DAMP (4‐diphenylacetoxy‐N‐methylpiperidine methiodide) the M3‐selective antagonist, was also the same as that of mammalian brains, i.e., 4‐DAMP>PZ>AF‐DX 116. The three insect brain receptors had 27–50‐fold lower affinity for PZ (Ki484–900 nM) than did the mammalian brain receptor (Ki16 nM), but similar to that reported for the muscarinic receptor subtype cloned fromDrosophila. Also, the affinity of insect receptors for 4‐DAMP (Ki18.9–56.6 nM) was much lower than that of the M3receptor, which predominates in rat submaxillary gland (Kiof 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, tho