Five subtypes of muscarinic acetylcholine receptors (m1-m5) have been expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system. Up to 6 nmol of muscarinic acetylcholine receptors were produced by 1 liter culture; 0.3 to 0.6 (human m1), 3 to 6 (human m2), 2 to 4 (rat m3), 1 to 2 (rat m4) and 0.5 to 1 (human m5) nmol. Pirenzepine, AF-DX116 and hexahidrosiladifenidol showed the highest affinity for the m1, m2 and m3 subtype, respectively, indicating that these receptors expressed in Sf9 cells retain the same substrate specificity as those in mammalian tissues or cultured cells. Among 32 kinds of muscarinic ligands examined in the present studies, prifinium was found to have the highest affinity for the m4 subtype, and pilocarpine, oxotremorine, McN-A343 and promethazine the highest affinity for the m5 subtype, although the differences in the affinities among the five subtypes were less than 10-fold. Alcuronium increased the binding of [3H]N-methylscopalamine to the m2 subtype, but not the m1, m4 and m5 subtypes and only slightly to the m3 subtype. Similar but smaller effects of fangchinoline and tetrandrine were found for [3H]N-methylscopalamine binding to only the m3 subtype. These effects may also be useful for the discrimination of individual subtypes.