The complete exon size and distribution pattern of the human alpha 5(IV) collagen gene COL4A5 has been determined. Seventeen genomic lambda phage clones, eight of which have been described previously (Zhou, J., Hostikka, S.L., Chow, L.T., and Tryggvason, K. (1991) Genomics 9, 1-9), spanning about 160 kilobases of DNA contained 140 kilobases of the gene itself. The clones covered the entire gene with the exception of exons 2 and 37 and their flanking regions so that the exact gene size could not be determined. The sequences of these two exons were, however, determined from polymerase chain reaction products. The COL4A5 gene has a structure highly homologous with that of COL4A1 which encodes the alpha 1(IV) chain. However, the COL4A5 gene contains 51 exons, or one less than COL4A1. The exon size pattern of the genes are similar, with 41 exons having identical sizes. All the exons were assigned to distinct EcoRI restriction fragments. The results may be useful for characterization of mutations in COL4A5 in patients with X chromosome-linked Alport syndrome.