Paroxysmal nocturnal hemoglobinuria is an acquired hemolytic anemia associated with somatic mutations in the X-linked gene PIG-A, which encodes a protein involved in the biosynthesis of glycosyl phosphatidylinositol anchors. To further elucidate the molecular basis of paroxysmal nocturnal hemoglobinuria, we have worked out a systematic and relatively rapid methodology to scan for mutations in the entire coding region of the PIG-A gene. By this methodology, we have identified 15 different somatic mutations in 12 patients. The mutations were spread throughout the entire PIG-A-coding region. Of the mutations, 10 caused frameshifts, 6 caused small deletions, 3 caused small insertions, and 1 caused deletion-insertion. Five single base pair substitutions caused three missense mutations, one nonsense mutation, and one defect in the donor splice site of intron 4. In each of 3 patients, two independent mutations were identified. The predominance of frameshift mutations may reflect selection for somatic mutations giving rise to clones with a completely nonfunctional PIG-A protein.