Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common red cell abnormalities, is characterized by a wide clinical, biochemical, and molecular heterogeneity. In this study we have determined the molecular basis of G6PD deficiency in a sample of 70 male subjects, originating from different parts of Italy, who all shared a clinical and biochemical phenotype identical or very similar to that of G6PD Mediterranean, the most common variant in Italy. In 59 cases (84%) we found the mutation 563 C --> T, previously known to be underlying the G6PD Mediterranean and the two polymorphic variants G6PD Cagliari and G6PD Sassari. From the remaining 11 we amplified the entire coding region of G6PD in 8 different fragments and subjected them to nonradioactive single-strand conformation analysis. Direct sequencing was then performed on abnormal fragments. By this approach we found six cases (8.5%) with 1360 G --> A mutation (G6PD Union) and two cases (2.8%) with 1376 G --> C (G6PD Cosenza). In the remaining three samples we found two other mutations: 1342 A --> G (two cases, 2.8%) and 1052 G --> T (one case, 1.4%). These two molecular defects have never been described before and were designated by us as G6PD S. Antioco and G6PD Partenope, respectively. Haplotype analysis suggested that all the non-Mediterranean mutations occurred independently on a normal G6PD allele. This study shows that the G6PD Union mutation has a high polymorphic frequency in the Italian population and that the genetic heterogeneity of G6PD Mediterranean-like variants is higher at the molecular level than expected from biochemical characterization.