Ligand-induced gene activation by nuclear receptors involves the recruitment of coactivators to hormone bound receptors. Recent results have shown that RAP250, also termed as ASC-2/PRIP/TRBP/NRC/AIB3, plays a critical role as a coactivator of nuclear receptors. In this study, we have determined the genomic organization of the human RAP250 gene in order to identify the promoter region. By searching the GenBank database for EST sequences, we could identify two previously unknown exons in the 5'-end of the gene. Our results show that the RAP250 gene consists of 15 exons spanning 111 kb. All sequences of the splice donor and acceptor sites fit the GT-AG role for splicing. We also show using linkage analysis that the mouse Rap250 gene is located on chromosome 2 close to the agouti gene. The RAP250 promoter is GC-rich and TATA-less, and contains multiple Sp1 binding sites and a MYC binding site. A reporter plasmid containing the 5' flanking region of the RAP250 gene showed significant activity compared to pGL3-basic and minimal thymidine kinase (TK) reporter plasmids in transfection experiments using luciferase reporter genes. Our data show that the RAP250 has a complex genomic structure with a promoter that is regulated by multiple transcription factors for its basal expression.