Erythrocyte (RBC) protein 4.2 (P4.2)-deficiency observed in Japanese individuals results in a hemolytic anemia associated with abnormally shaped (spherocytic, ovalocytic, and elliptocytic), osmotically fragile RBCs, the clinical presentation of which resembles hereditary spherocytosis (HS). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, P4.2-deficient individuals contain less than 1% of the normal membrane content of P4.2 and immunologic analysis shows that the P4.2 present exists as an equimolar doublet of 74-Kd and 72-Kd bands, in contrast to normal RBC membranes where a discrete 74-Kd band is not observed. RBC membranes from both of the biologic parents of a P4.2- deficient individual contained both the 74-Kd and the 72-Kd bands, demonstrating their heterozygosity for the P4.2 defect. The molecular basis of Japanese P4.2-deficiency was investigated by reverse transcription of total reticulocyte RNA, followed by polymerase chain reaction (PCR) amplification, subcloning, and sequencing. The complete cDNA sequence of a P4.2-deficient patient showed a single point mutation that changes codon 142 from GCT (alanine) to ACT (threonine) (Protein 4.2NIPPON). The mutation also eliminated an HgaI restriction site, therefore allowing rapid screening for the presence of the mutation. Screening of PCR-amplified genomic DNA showed that the mutation was present in the homozygous state in four (eight chromosomes) unrelated Japanese P4.2-deficient individuals and absent in 35 (70 chromosomes) P4.2-normal controls (including 15 Japanese [30 chromosomes]). The presence of the mutation was confirmed by allele- specific hybridization. The mutation occurred in an alternatively spliced exon that is present in two of four P4.2 mRNA splicing isoforms. These results demonstrate that Japanese P4.2-deficiency is closely associated with the P4.2 gene and does not arise secondarily to a defect in another membrane protein, and further suggest that the P4.2- deficiency is related to the pathogenesis of the hemolytic anemia in this variant form of recessively inherited spherocytosis.