Mutations of WASP (Wiskott-Aldrich syndrome protein) underlie the severe immunodeficiency/platelet disorder Wiskott-Aldrich syndrome (WAS) and its milder variant X-linked thrombocytopenia (XLT). The affected gene, a 12-exon structure on the X-chromosome, is expressed exclusively in blood cells. The encoded product WASP is a 502-amino-acid scaffolding protein that functions in stimulus-induced nucleation of actin filaments to form dynamic cell surface projections. To date, more than 150 mutations have been identified in 300 WAS/XLT kindred worldwide, generally through methodologies that include sophisticated exon screening steps such as single-strand conformation analysis. We report here a simpler protocol, which was designed for use in clinical settings to identify the mutations of newly diagnosed patients. The approach relies on directly sequencing amplified exons according to a staggered schedule based on statistical evaluation of previous cases. In a 2 1/2-year trial, samples from 28 consecutive patients were analyzed; these included 3 "blindly labeled" previously studied cases. The mutations that were identified include a broad spectrum (8 missense, 3 nonsense, 5 splice site mutations, 11 small insertion/deletions, 1 large deletion) and were broadly distributed (in 10 of the 12 exons). All mutations were verified and no discrepancies were encountered. Per patient, a mean of six DNA sequencing reactions and 6-7 h of staff effort sufficed for mutation identification and verification, indicating that the protocol is cost-effective. This cumulative experience demonstrates the suitability, reliability, and versatility of the new protocol.