The beta4 subunit is a component of the neuronal nicotinic acetylcholine receptors which control catecholamine secretion in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was characterized. A proximal region (from minus sign99 to minus sign64) was responsible for the transcriptional activity observed in chromaffin, C2C12, and COS cells. Within this region two cis-acting elements that bind transcription factors Sp1 and NF-Y were identified. Mutagenesis of the two elements indicated that they cooperate for the basal transcription activity of the promoter. The human beta4 promoter, that was also characterized, shared structural and functional homologies with the bovine promoter. Thus, two adjacent binding elements for Sp1 and NF-Y were detected. Whereas the Sp1 site was an important determinant of the promoter activity, the NF-Y site may have cell-specific effects. Given that these promoters showed no structural or functional homology with the previously characterized rat beta4 subunit promoter (Bigger, C. B., Casanova, E. A., and Gardner, P. D. (1996) J. Biol. Chem. 271, 32842--32848) except for the involvement of an Sp1 binding element, we propose that constitutive expression of the beta4 subunit gene in these three close species may be controlled by the general transcription factor Sp1. Nevertheless, other components could determine species-specific beta4 subunit expression.