To develop a general method for analysis of the mutation and prenatal diagnosis of X-linked hyper-IgM syndrome (XHM), the human CD40 ligand (hCD40L) gene was cloned and sequenced with special reference to the 5' and 3' flanking regions and exon/intron boundaries. The hCD40L gene consists of five exons and four introns, as already reported by others. Two major transcription initiation sites were identified at 67 bp and 64 bp upstream from the ATG initiation codon. The hCD40L mRNA transcripts terminated at 321 bp, 327 bp and 987 bp downstream from the TGA stop codon. Based on the intronic sequences, oligonucleotide primers were designed for amplifying the coding region of each exon separately. Polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis was successfully applied to screening for the defective hCD40L gene in a family with XHM. The nonsense mutation, Trp140 (TGG)-->stop (TAG) in exon 5, was found in the mother and an affected child. We also performed prenatal diagnosis by PCR-SSCP during the first trimester of pregnancy in this family.