The M₂ muscarinic acetylcholine receptor mutant (M, mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein NEP) at its N-terminus and expressed in Escherichia coli. The expression level was 0.2 urnol receptor per 100 ml culture, as assessed as [³H] L-quinuclidinyl benzilate ([³H] QNB) binding activity, when the BL 21 strain was cultured at 37°C to a late growth phase and the expression was induced by isopropyl β-thiogalactoside at 20°C. No [³H]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37°C instead of 20°C. The MBP-M₂ mutant expressed in E. coli showed the same ligand binding activity as the M₂ mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [³H] QNB with carbamylcholine and atropine. The MBP M₂ mutant was solubilized, purified with Co²⁺-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein G_o or G_il in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [³⁵S] GTPγS binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [³⁵S]GTPγS binding were the same as those observed for the M₂, mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol. These results indicate that the MBP-M₂ mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M₂ mutant expressed in Sf9, and that cholesterol is not essential for the function of the M₂ muscarinic receptor.