The human ovalbumin (ov) serpins are associated with tumorigenesis, inflammation, and protection from autolysis by granule proteinases. Their genes are located at 18q21 or 6p25, falling into two structurally very similar but distinct categories depending on the presence or absence of a particular exon. Analysis of ov-serpin gene structure provides an opportunity to elucidate the mechanisms contributing to the formation of the larger serpin gene superfamily. Here we have identified a new gene (PI8L1) at 6p25 that is 72% identical to the 18q21 gene PI8. FISH analysis using the 3′ untranslated region of PI8 yielded an additional signal at 18q23, separable from the known 18q21.3 signal by the t(1;18)(p32;q23) chromosomal translocation. The presence of more than one PI8-related gene was confirmed by analysis of human genomic DNA using the same probe. Cloning and analysis of PI8 showed that its intron number and phasing are identical to those of the 6p25 genes PI6, PI9, and ELANH2, and it lacks the interhelical variable loop exon found in other 18q21 genes. PCR analysis demonstrated that PI5 at 18q21 also lacks this exon, indicating that it is organized identically to the 6p25 genes. By contrast, PI10 and megsin have this exon and resemble the other 18q21 genes, PLANH2, SCCA-1, and SCCA-2, in structure. Using these data with an ov-serpin phylogenic tree we have constructed, we propose that the ov-serpin gene clusters arose via interchromosomal duplication of PI5 (or a precursor) to 6p25, followed by duplication at 6p25, and a more recent interchromosomal duplication from 6p25 to 18q to yield PI8.