cDNAs encoding for five mAChR subtypes (M1-M5) were cloned under different promoters in various eukaryotic vectors and each subtype was expressed in different mammalian cell lines. CHO-K1 cell line was the best for generating stable cell lines expressing muscarinic receptors. Immunofluorescence and flow cytometry revealed that expression of M1-M5 was primarily localized on the cell membrane. Western blotting and radio-ligand binding studies revealed that expression of each receptor was stable at higher passages.