Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) variants was carried out in 150 unrelated G6PD deficient blood donors from the region of Campinas, Brazil. By allele specific oligomer hybridization or digestion of exon 4 of the G6PD gene with the restriction endonuclease Nla III, we detected the 202 G→A mutation in 146 individuals. This mutation was associated with the 376 G→A substitution and only one haplotype was observed in these individuals. Digestion of exon 6 with the restriction enzyme Mbo ll showed the presence of the Mediterranean variant in three individuals. Haplotype analysis showed, in all three samples, a T at nt 1311 and the C at nt 13 in intron 11 suggesting a European origin of this variant. By SSCP analysis and direct sequencing we detected the mutation nt 1003 G→A (335 Ala→Thr) in one blood donor. This mutation was previously described in a boy of Indian ancestry and the variant was denominated G6PD Chatam. The case described here has no Indian ancestry; thus, we presume that the mutations have arisen independently, although we do not know the haplotype of the Indian patient. The haplotype of our case was the most common observed in our population (Pvu ll, Bsp Hl+, Pstl+, 1311C, Nla III-). Thus, our data indicate that G6PD A- with the 202 G→A mutation is the most frequent G6PD deficiency in the population of southeastern Brazil. The remaining variants had a Mediterranean origin. These results are in agreement with the origin of the Brazilian population.