Treatment of neuronal cells with leukemia inhibitory factor (LIF) results in increased M(2) muscarinic acetylcholine receptor promoter activity. We demonstrate here that multiple promoter elements mediate LIF stimulation of M(2) gene transcription. We identify a LIF inducible element (LIE) in the M(2) promoter with high homology to a cytokine-inducible ACTG-containing sequence in the vasoactive intestinal peptide promoter. Mutagenesis of both a STAT (signal transducers and activators of transcription) element and the LIE in the M(2) promoter is required to attenuate stimulation of M(2) promoter activity by LIF completely. Mobility shift assays indicate that a LIF-stimulated complex binds to a 70 base pair M(2) promoter fragment. Furthermore, a STAT element within this fragment can bind to LIF-stimulated nuclear STAT1 homodimers in vitro. Mutagenesis experiments show that cytokine-stimulated activation of M(2) promoter activity requires tyrosine residues on glycoprotein 130 (gp130) that are also required for both STAT1 and STAT3 activation. Dominant negative STAT1 or STAT3 can block LIF-stimulated M(2) promoter activity. Real-time RT-PCR analysis indicates that LIF-stimulated induction of M(2) mRNA is partially dependent on protein synthesis. These results show that regulation of M(2) gene transcription in neuronal cells by LIF occurs through a complex novel mechanism that is dependent on LIE, STAT and de novo protein synthesis.