The human genes encoding the "ovalbumin" subgroup of closely related serine proteinase inhibitors (serpins) are located at 18q21.3 and 6p25. Those at 6p25 include proteinase inhibitor 6 (PI-6; gene symbol PI6), proteinase inhibitor 9 (PI-9; gene symbol PI9) and monocyte neutrophil elastase inhibitor (M/NEI; gene symbol ELANH2). Here we describe the fine mapping of these genes to a 200-kb region of chromosome 6 that includes the markers WI-8835 and D6S1338, and the establishment of the gene order: tel-PI6-PI9-ELANH2-cen. PI6 and ELANH2 are transcribed towards the telomere, and structural analysis shows that PI6 and PI9 are organized identically, having seven exons and six introns. PI6 and PI9 are almost identical in structure to the ovalbumin serpin genes at 18q21.3. The 18q21.3 genes have an extra exon and intron, otherwise all the other exon/intron boundaries are conserved between the two groups. These results represent the first detailed map of the chromosome 6 serpin gene cluster, and demonstrate that although they are very closely related, the 6p25 and 18q21-->q23 ovalbumin serpin genes form two structurally distinct groups. These findings do not support a previously proposed model for evolution of the clusters which invoked an inter-chromosomal duplication of the entire 6p25 group to 18q21.3.