We have determined the exon-intron structure of the 3' half of the gene for the human type IV collagen alpha 5 chain that is affected in X-chromosome-linked Alport syndrome. Six overlapping lambda phage genomic clones containing exons 1-14 (as counted from the 3' end) and two additional overlapping genomic clones containing exons 16-19 spanned a total of 60 kb, 9.5 kb of which were the 3' flanking region. The exon-intron structure was elucidated by restriction enzyme mapping, nucleotide sequencing, and heteroduplex analyses. The sequences of all of the 19 most 3' exons and their flanking sequences were determined from the genomic clones, with the exception of exon 15, which was sequenced after amplification from genomic DNA with the polymerase chain reaction. The results show that the genes for the alpha 5(IV) and alpha 1(IV) chains have an almost identical exon size pattern in the 3' half. In contrast, there is not a clear conservation of intron sizes between the two genes, although both genes may have a similar total size. The current results have allowed the identification of three mutations in the alpha 5(IV) gene in three kindreds with Alport syndrome, and the gene structure and sequencing data presented should facilitate the analysis of other as yet unidentified mutations in this heterogeneous genetic disease.