The alpha5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from -111 to -40). Deletion analysis and transient transfections showed that a 266-base pair region (-111 to +155) gave rise to approximately 77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the alpha5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased alpha5 promoter activity by approximately 50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50-69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment -111 to -27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in alpha5 subunit expression, as has also been demonstrated previously for alpha3 and beta4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits.