Genomic and cDNA clones encoding a muscarinic acetylcholine receptor from Drosophila melanogaster have been isolated. Sequence analysis demonstrates that this gene encodes a receptor with a high degree of amino acid identity to the mammalian muscarinic acetylcholine receptors and has three introns in the portion of the gene encoding the third putative cytoplasmic loop. A full-length cDNA clone has been placed under the control of the mouse metallothionein promotor and transfected into mouse Y1 adrenal cells. The receptor expressed in these cells exhibits the high-affinity binding for the antagonists quinuclidinyl benzilate and atropine expected of a muscarinic receptor. The Drosophila muscarinic receptor, when expressed in Y1 cells, is physiologically active, as measured by agonist-dependent stimulation of phosphatidylinositol metabolism.