ApolipoproteinB (apoB) mRNA editing involves a C to U deamination of the nuclear apoB mRNA and occurs in mammalian small intestine and in the liver of certain species. This reaction is mediated by a multicomponent enzyme complex that includes a catalytic subunit, apobec-1. Apobec-1 mRNA is widely expressed in the rat and mouse and is subject to tissue-specific regulation. In order to understand the basis for the species- and tissue-specific pattern of apobec-1 gene expression we have cloned and characterized the rat chromosomal apobec-1 gene. We demonstrate its structural organization and regulation in comparison to that of the mouse apobec-1 gene. The rat apobec-1 gene spans 16 kb and includes one untranslated (exon A) and five translated exons (exons 1-5). The mouse apobec-1 gene contains eight exons, of which the first three (exons A, B, C) are untranslated. Independent approaches demonstrated three distinct clusters of transcription initiation sites in both species, including exon A, the distal region of exon 1, and a separate group in the proximal region of exon 1. These transcription start sites generate three distinct mRNA species whose proportions differ in a tissue-specific fashion. Promoter-luciferase reporter constructions using regions flanking exon A and exon 1 of the rat apobec-1 gene identified two functional regions upstream of exon 1 that independently promote luciferase expression in transfected hepatoma and colon cancer cells. These data serve as a basis for an understanding of the regulation of apobec-1 gene expression, in particular the mechanisms that serve to restrict its expression to the gastrointestinal tract in higher mammals.