We have isolated the gene encoding the neuronal intermediate filament protein alpha-internexin using low stringency hybridization conditions and an NF-M (neurofilament middle molecular weight subunit) cDNA probe. This gene consists of three exons and two introns. The sequence data and the exon-intron organization of the gene establish its classification as a type IV intermediate filament gene. Transient transfection experiments showed that up to 5 kilobases of the alpha-internexin promoter region transcribed equally well in both the alpha-internexin-expressing and -nonexpressing cell lines. The results also demonstrated that the region from -77 to +73 relative to the transcription start site was sufficient for accurate basal transcription, but inclusion of the -254 to -78 region was required for efficient transcription. Sequence analysis shows that the -254 to -78 region contains several potential positive regulatory elements.