Paroxysmal nocturnal hemoglobinuria (PNH) may arise during long-term follow- up of aplastic anemia (AA), and many AA patients have minor glycosylphosphatidylinositol (GPI) anchor-deficient clones, even at presentation. PIG-A gene mutations in AA/PNH and hemolytic PNH are thought to be similar, but studies on AA/PNH have been limited to individual cases and a few small series. We have studied a large series of AA patients with a GPI anchor-deficient clone (AA/PNH), including patients with minor clones, to determine whether their pattern of PIG-A mutations was identical to the reported spectrum in hemolytic PNH. AA patients with GPI anchor-deficient clones were identified by flow cytometry and minor clones were enriched by immunomagnetic selection. A variety of methods was used to analyze PIG-A mutations, and 57 mutations were identified in 40 patients. The majority were similar to those commonly reported, but insertions in the range of 30 to 88 bp, due to tandem duplication of PIG-A sequences, and deletions of more than 10 bp were also seen. In 3 patients we identified identical 5-bp deletions by conventional methods. This prompted the design of mutation-specific polymerase chain reaction (PCR) primers, which were used to demonstrate the presence of the same mutation in an additional 12 patients, identifying this as a mutational hot spot in the PIG-A gene. Multiple PIG-A mutations have been reported in 10% to 20% of PNH patients. Our results suggest that the large majority of AA/PNH patients have multiple mutations. These data may suggest a process of hypermutation in the PIG-A gene in AA stem cells.