Abstract : Five subtypes of the muscarinic receptor have been cloned from both the rat and human genomes. Although all five genes have the coding sequences in a single exon, their structures 5′ of the initiation codon are largely uncharacterized, except for the M4 receptor. In the brain, muscarinic receptors mediate motor and memory function by interaction with their ligand acetylcholine. In addition, the M1 muscarinic subtype has been implicated in behavior, stress‐adaptive cardiovascular reflexes, and blood pressure regulation. In the current study the M1 muscarinic receptor noncoding 5′‐flanking region has been identified and characterized, including the promoter and two 5′ noncoding exons located ~13‐14 kb from the coding exon. Similar to the M4 muscarinic receptor gene the M1 promoter is GC‐rich, contains no TATA box, but has two potential CAAT boxes and several putative binding sites for transcription factors such as SP1 and AP‐1‐3. The transcription initiation site was identified by RNase protection and primer extension. Promoter activity was confirmed in transient expression assays, using luciferase reporter constructs. A 0.89‐kb fragment consisting of 480 bp of the promoter, exon 1, and part of intron 1 expressed luciferase activity in two M1 receptor‐expressing cell lines (CCL‐107 and CCL‐147), whereas a longer fragment (1.5 kb) that extends into intron 2 demonstrated significantly increased luciferase activity. The constructs exhibited responses indicating the presence of functional glucocorticoid‐, acute‐phase‐, and heat shock‐responsive elements.