Fanconi anemia (FA) is an autosomal recessive disorder exhibiting chromosomal fragility, bone-marrow failure, congenital abnormalities, and cancer. At least eight complementation groups have been described, with group A accounting for 60%-65% of FA patients. Mutation screening of the group A gene (FANCA) is complicated by its highly interrupted genomic structure and heterogeneous mutation spectrum. Recent reports of several large deletions of FANCA, coupled with modest mutation-detection rates, led us to investigate whether many deletions might occur in the heterozygous state and thus fail to be detected by current screening protocols. We used a two-step screening strategy, in which small mutations were detected by fluorescent chemical cleavage of the FANCA transcript, and heterozygosity for gross deletions was detected by quantitative fluorescent multiplex PCR. We screened 26 cell lines from FA complementation group A for FANCA mutations and detected 33 different mutations, 23 of which were novel. Mutations were observed in all 26 cell lines and included 43 of a possible 52 mutant alleles (83%). Of the mutant alleles, 40% were large intragenic deletions that removed up to 31 exons from the gene, indicating that this may be the most prevalent form of mutation in FANCA. Several common deletion breakpoints were observed, and there was a highly significant correlation between the number of breakpoints detected in a given intron and the number of Alu repeats that it contained, which suggests that Alu-mediated recombination may explain the high prevalence of deletions in FANCA. The dual screening strategy that we describe may be useful for mutation screening in other genetic disorders in which mutation-detection rates are unexpectedly low.