The Duffy gene has been shown not to be split by introns, even in its 5′ untranslated region, and to be expressed not only in erythroid but in postcapillary venule endothelium of almost every organ in the body. To further investigate the transcriptional start position in erythroid and postcapillary venule endothelium, we performed 5′-rapid amplification of cDNA ends (5′-RACE). While every positive clone of 5′- RACE encoded the identical sequence of previously identified cDNA downstream from nucleotide 203, the upstream sequences were different. The upstream sequences corresponded to the sequence from nucleotide - 279 to -308/-357 in erythroblasts and from -279 to -355/-383 in lung and were regarded as comprising a novel exon. This novel exon encoded seven residues initiated with a methionine, linked to nucleotide 203 in- frame and in agreement with the GT-AG splicing rule. The major erythroid transcriptional start position was identified in human erythroleukemia cells by primer extension and in bone marrow by ribonuclease protection analysis at 34 bases upstream from the first ATG codon. Distinctively, in lung and kidney, the transcription was started at 82 bases upstream from the ATG. Both Northern blotting and reverse transcription-polymerase chain reaction followed by Southern analysis indicated a predominance of the novel spliced form of mRNA of about 50- to 200-fold comparing with the unspliced form, in every studied organ and erythroid lineage cells. The spliced form of cDNA has been transfected into a human erythroleukemic cell line, K562, and the expressed protein reacted with Duffy-specific murine monoclonal antibody Fy6. These studies indicate that the product from the spliced form of mRNA is the major product of the Duffy gene in the erythroid lineage and postcapillary venule endothelium.