The Duffy blood group antigen has been characterized by its roles on red blood cells: as a receptor for the malarial parasites and as a promiscuous receptor for chemokine superfamily. Recently, the Duffy blood group associated glycoprotein D (gpFy) cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). In this report we describe the organization of genomic DNA coding for the gpFy and elucidate the molecular nature of Fya/b polymorphisms. By a Southern blotting analysis probed with gpFy cDNA, gpFy gene was shown to be composed of three DNA fragments; 1.1-kb Sac I, 1.9-kb EcoRI, and their intervening 47-bp fragments. We cloned the 1.1-kb Sac I and 1.9- kb EcoRI fragments by inverted polymerase chain reaction (IPCR) procedure. The promoter region of the gpFy gene was cloned by IPCR of 1.1-kb Sac I fragment and the 3′ flanking sequence was cloned by IPCR of 1.9 kb EcoRI fragment. The both IPCR products contained on both side the known gpFy cDNA sequence without introns, as expected. Although no TATA or CCAAT boxes are present in the promoter sequence, several transcription factor binding site motifs are contained, including AP-1, HNF-5, TCF-1, ApoE B2, W-element, H-APF-1, and Sp-1. The 3′ flanking region has two additional polyadenylation signals, other than that used in the cDNA, and also has an indirect and a direct repeat sequence clustered with the 5′ flanking region. These facts indicate a possibility that the gpFy gene has been evolved by multiple retrotransposition events. By comparing the coding area of the gpFy gene in 28 Duffy-positive individuals, we elucidated that one base change that results in an amino acid substitution [GA-T(Asp44)-- >GGT(Gly)] is in accordance with the Fya/Fyb polymorphism. This fact proves that the gpFy cDNA and its gene described in this report encode the Duffy blood group system.