Mutations in the gene for Bruton's tyrosine kinase (Btk) are responsible for X-linked agammaglobulinemia (XLA). Thus far, mutations in this gene have been identified based on alterations in Southern or Northern blot analysis or cDNA sequence. To permit detection of mutations in genomic DNA, we designed PCR primers to flank each of the 19 exons of Btk with splice sites. Two overlapping PCR products were employed for exons longer than 230 base pairs. Single strand conformation polymorphism (SSCP) analysis was used to screen PCR products from 30 unrelated families presumed to carry a Btk mutation. It was possible to amplify DNA in every reaction from every patient, indicating that large deletions in Btk are uncommon. Twenty three different mutations were found in 25 unrelated families, including one family in whom DNA was available from a carrier but not an affected patient. Seven mutations were single base pair substitutions resulting in premature stop codons scattered throughout the gene. Small insertions or deletions causing frameshifts and secondary premature stop codons constituted an additional seven mutations. One patient had a point mutation in the start codon and one patient had a mutation in a splice donor site. Point mutations resulting in amino acid substitutions were seen in nine patients. Northern blot analysis of RNA from three patients with premature stop codons showed an absence of Btk transcript whereas four patients with amino acid substitutions had normal amounts of transcript of normal size. These studies document the considerable variability in the Btk mutations causing XLA and they demonstrate an approach that will be useful for carrier detection as well as mutation identification.