We cloned and characterized the mouse gene for microtubule-associated protein (MAP) 1A, an important protein for neuronal morphology and mitotic spindle formation. We also investigated the 5' untranslated region of the gene to characterize the promoter units. Two alternative transcripts different in the 5' region were identified by 5' RACE. Both transcripts were principally observed in the brain. Genomic cloning revealed that exons 1, 2, and 4 generate the 5' part of a long transcript, whereas exons 3 and 4 generate a short transcript. Putative 5' and intronic promoters flanking exons 1 and 3, respectively, are GC-rich and lack a canonical TATA box. DNase I footprinting from mouse cells revealed that several potential cis-elements were occupied by nuclear proteins. A reporter assay system in conjunction with a number of deletion and mutation constructs was used to test the two putative promoters. Both putative promoters showed transactivity and their function was dependent upon Sp1 sites. In addition, an NF-1 site, an HNF3B site, and an AP-1/ATF site were necessary for basal promoter activity of the intronic promoter. Our data provide insight into the regulatory mechanisms that govern the expression of the MAP1A gene.