The COL4A5 gene from 40 patients with Alport's syndrome was examined using single-strand conformation substitution at the acceptor site (-2) of intron 50 and a G-to-C substitution at the donor site (+1) of intron 47, respectively. The transcript in peripheral leukocytes from the former had a 10-nucleotide deletion. This shortened transcript was derived from abnormal splicing in a cryptic acceptor site within exon 51. This could be translated into a protein with an alteration of three amino acids followed by premature termination, which eliminated 23 amino acids from the carboxyl end. Gene tracking revealed that the mother and a brother carried the mutant allele. In the latter, the transcript in leukocytes was normal, but that in cultured skin fibroblasts showed skipping of exon 47, the result being that 71 amino acids were absent. Glomerular basement membrane from the patient did not react with the anti-alpha 5(IV) antibody. His maternal grandmother, mother, and a sister, all with abnormal urinalysis, carried the mutant allele. Thus, the appearance of exons of the COL4A5 gene in leukocytes may differ from that in fibroblasts. If kidney mRNA is not available, mRNAs from cultured skin fibroblasts, in addition to leukocytes, can be used for gene analysis in subjects with Alport's syndrome.