The nuclear factor 1 (NF1) protein family functions as a cellular transcription factor as well as an adenovirus DNA replication factor. This family consists of four subtypes, NFI-A, NFI-B, NFI-C, and NFI-X, each encoded by a different gene. Each subtype possesses different isoforms generated by alternative splicing. To date, only a porcine NFI-C gene has been cloned, and the gene structures of the other NF1 proteins have not yet been identified. We recently isolated four kinds of NFI-A cDNA clones from the rat liver. To gain additional insight into the structure of NFI-A, we isolated the rat NFI-A gene. This gene is composed of 11 exons spanning over 70 kb. All of the exon/intron boundaries are consistent with the GT/AG rule, and consensus sequences surrounding the splice boundaries are also found. The 5'-flanking region lacks a canonical TATA box, but contains several GC-box and AP2 binding sites. A 5'-rapid amplification of cDNA end analysis indicated that the transcription of the NFI-A gene is initiated at multiple sites. We also found conservation in the genomic structure between the rat NFI-A and the porcine NFI-C, suggesting that duplication of an ancestral gene occurred rather recently to produce the NFI-A and NFI-C genes.