Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, and radiation sensitivity. The responsible gene, ATM, has an extensive genomic structure and encodes a large transcript with a 9.2 kb open reading frame (ORF). A-T mutations are extremely variable and most of them are private. We streamlined a high throughput protocol for the search for ATM mutations. The entire ATM ORF is amplified in a single RT-PCR step requiring a minimal amount of RNA. The product can serve for numerous nested PCRs in which overlapping portions of the ORF are further amplified and subjected to restriction endonuclease fingerprinting (REF) analysis. Splicing errors are readily detectable during the initial amplification of each portion. Using this protocol, we identified 5 novel A-T mutations and completed the elucidation of the molecular basis of A-T in the Israeli population.