Cholinergic muscarinic receptor genes are members of the G-protein receptor gene superfamily. In this study we describe the structure of the gene and promoter of the rat m4 muscarinic receptor gene. A rat cosmid clone containing the coding region for the m4 gene and 25 kilobases of upstream sequence was isolated. This clone directed expression of the rat m4 gene when introduced in IMR32 cells, a human neuroblastoma that expresses m4, but did not drive expression when introduced into Chinese hamster ovary cells, a line that does not express the m4 gene. S1 nuclease, modified 5'-rapid amplification of cDNA ends and polymerase chain reaction analysis of rat cosmid DNA and cDNA showed that the gene consists of a 2.6-kilobase coding exon, extending 34 base pairs (bp) upstream from the initiating ATG, separated from a 460-493 bp noncoding exon by a 4.8-kilobase intron. DNA sequence analysis shows that the non-coding exon is GC-rich and that the promoter does not contain a TATA or CAAT box and has several consensus sequences for enhancer elements including five Sp-1 binding sites, one AP-2 site, one AP-3 binding site and two E-boxes within the proximal 600 bp. A reporter construct consisting of 1440 bp of flanking DNA and 80 bp of the first exon cloned into a luciferase reporter plasmid, drove cell specific expression in transient transfection assays. Removal of 1088 bp of the 5' end of this construct resulted in expression in non-m4 expressing cell lines suggesting there is a repressor element in this region.