Among over 50 distinct mutations causing glucose-6-phosphate dehydrogenase (G6PD) deficiency, only two deletion mutations have so far been reported. Using nonradioisotopic single-strand conformation polymorphism analysis, we found two additional deletion mutations in two Japanese G6PD-deficient patients with nonspherocytic hemolytic anemia. Case no. 1 had a 3-nucleotide deletion in exon 6 predicting a deletion of a serine at amino acid 188 or 189, which caused a class 1 variant G6PD Tsukui. Case no. 2 had a 3-nucleotide deletion in exon 5 predicting a deletion of a lysine at residue 95, which caused a class 2 variant G6PD Urayasu. The 188th serine, which might be deleted in G6PD Tsukui, is located close to the putative G6P binding site. The 188th serine is also involved in the amino acid substitution in G6PD Mediterranean, but the kinetics of these two variants are totally different. The residue with an amino acid deletion in G6PD Urayasu was distant from the substrate binding sites and was located in a region with low sequence homology among species. The different properties of variants having mutations in exons 5 and 6 suggest that these two exons code distinct functional domains of the enzyme.