The present epidemiological study of the molecular characteristics of beta-thalassemia in Sicily was prompted by the disparate phenotypic expression (in clinical status and absolute HbF level) observed in two beta-thalassemic homozygotes who were also homozygous for the beta-like globin gene cluster haplotype III. We suspected that polymorphisms within haplotype III could be the cause for the discrepancy. Based on the association of particular conformations of the (AT)xT(y) motif (-540 5' to the beta gene) with milder forms of thalassemia and sickle cell anemia, 38 homozygous beta-thalassemia patients were studied to define their haplotypes, the -158 site 5' to the G gamma gene (linked to haplotype III) and the structure of the (AT)xT(y) motif. We found that the patient who was phenotypically mild and homozygous for beta-thalassemia, haplotype III, and the -158 C----T mutation was homozygous for the rare (AT)9T5 motif. In contrast, the patient homozygous for beta-thalassemia, haplotype III, and the -158 mutation, but exhibiting a severe clinical course, was homozygous for the (AT)7T7 configuration. Others have suggested that (AT)9T5 is a negative regulatory protein binding sequence, and it is a silent carrier state for beta-thalassemia. The usual configuration (AT)7T7, has considerably less affinity for regulatory protein binding, and it is the most common configuration in Sicilian beta-thalassemics (67 of the 78 chromosomes studied). Within the 38 patients studied, seven were informative because they had various combinations of the (AT)9T5 and (AT)7T7 motif, and the -158 C----T mutation. The results in these patients suggest that only the co-presence of the (AT)9T5 configuration and a C----T change at -158 5' to the G gamma gene is associated with high HbF expression and a mild clinical phenotype. We postulate that these two regions of the beta-like globin gene cluster interact, when endowed with the proper sequences, to enhance the expression of HbF secondary to anemia.