Mutation screening of the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare non-truncating sequence variants in the BRCA1 and BRCA2 genes is problematic because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. The BRCA1 331G > A substitution mutation, which occurs at the last nucleotide of exon 5, results in an Arg-to-Lys change at codon 71 (R71K). cDNA analysis indicated that the R71K mutation significantly increases the level of a transcript, characterized by a 22 bp deletion in exon 5, which putatively produces a truncated BRCA1 protein of 63 amino acids. The mutation completely abolishes normal splicing as the mutant allele does not generate any normal transcript. Analysis of a tumor specimen indicates loss of heterozygosity. These results support the conclusion that BRCA1 331G > A (R71K) is a deleterious mutation.