Muscarinic acetylcholine receptor genes are members of the G-protein coupled receptor superfamily. Each member of this family studied to date appears to have a distinct expression profile, however the mechanisms determining these expression patterns remain largely unknown. We have previously isolated a genomic clone containing the M 1 muscarinic receptor gene and determined its gene structure [Pepitoni, Wood and Buckley (1997) J. Biol. Chem. 272 , 17112-17117]. We have now identified DNA elements responsible for driving cell specific expression in transient transfection assays of immortalized cell lines. A region of the gene spanning 974 nucleotides and containing 602 nucleotides of the first exon is sufficient to drive specific expression in cell lines. Like the M 4 and M 2 gene promoters, the M 1 promoter contains an Sp1 motif which can recruit transcription factor Sp1 and at least one other protein, although this site does not appear to be functionally important for M 1 expression in our assay. We have identified a region within the first exon of the M 1 gene that regulates expression in cell lines, contains several positive and negative acting elements and is able to drive expression of a heterologous promoter. A polypyrimidine/polypurine tract and a sequence conserved between M 1 genes of various species act in concert to enhance M 1 transcription and are able to activate a heterologous promoter. We show that DNA binding proteins interact in vitro with single-stranded DNA derived from these regions and suggest that topology of the DNA is important for regulation of M 1 expression.