We have screened 110 unrelated Alport syndrome kindreds for mutations in the exon 48 region of the COL4A5 collagen gene. Denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified region of exon 48 revealed sequence variants in DNA from affected males and carriers of three unrelated kindreds. All three kindreds have classical Alport syndrome of the juvenile type. DNA-sequencing analyses demonstrated two different single base changes in the codon for arginine-1563 located in exon 48. In Utah kindred 2103, there was a substitution of C by T resulting in the change of the CGA codon for arginine to the translation stop codon TGA. In Utah kindred 2123 and in the Danish kindred A13, there was a C-->T mutation in the noncoding strand changing the same codon to CAA for glutamine. Both mutations were confirmed by allele-specific hybridization on PCR-amplified DNA from other family members.