Glucose-6-phosphate dehydrogenase (G6PD); EC 1.1.1.49 from thirty-six unrelated Spanish males was partially purified from blood, and the variants were characterized biochemically and electrophoretically according to the methods recommended by the world Health Organization. Subjects were from multiple geographic regions within Spain, and all suffered from hemolytic anemia, either acute (34 cases) or chronic nonspherocytic (2 cases). Almost all the variants studied presented residual erythrocyte G6PD activity ranging from 0 to 10% of normal, and five different mutants were responsible for the deficient phenotype. Three variants were similar to others previously described: G6PD Mediterranean (11 cases), G6PD Athens-like (3 cases), and G6PD Union (2 cases). The remaining variants were different from the numerous variants already reported and have been considered as new mutants. Provisionally they are called G6PD Betica (19 cases) and G6PD Menorca (1 case). The present study constitutes the first attempt to characterize the deficient G6PD variants found in Spain and supplies new data on the relationship between molecular characteristics of deficient variants and their clinical manifestations. The most important findings can be summarized as follows: (1) The Spanish population is characterized by an important heterogeneity in G6PD deficiency. (2) Although G6PD Mediterranean is very frequent, it presents a relatively high degree of polymorphism. (3) Favism has been observed associated with all kinds of variants described here. (4) G6PD Betica, which is the most frequent variant found in subjects of Southern Spanish origin, has been observed associated with favism in all cases except one.