Patients from the eastern province of Saudi Arabia who have sickle cell anemia have high circulating levels of fetal hemoglobin (hemoglobin F, 17 percent), and they therefore have a mild form of the disease. To examine the molecular basis of the elevated production of hemoglobin F, we searched for mutations in the promoter regions of the two hemoglobin F gamma-globin genes (G gamma and A gamma). The DNA sequences 450 bp (base pairs) upstream of both the G gamma and A gamma globin genes were normal except for a single-base cytosine-to-thymidine (C----T) substitution at -158 bp 5' to the cap (preinitiation) site of the G gamma-globin gene of the high-hemoglobin-F chromosome. We searched for an association between this -158 C----T substitution and the production of hemoglobin F and G gamma in normal Saudis and Saudis with sickle cell disease or trait. The substitution was present in nearly 100 percent of the patients with sickle cell disease or trait, and in 22 percent of the normal Saudis. Homozygosity for this mutation had no demonstrable effect on hemoglobin F production in the normal Saudi population. We conclude that this mutation is not uniquely responsible for the increase in hemoglobin F in Saudi patients. It may nevertheless have an important role in regulating hemoglobin F production, but its expression is complex and requires interaction with additional factors, such as hemolytic stress or other molecular determinants, possibly linked to the sickle cell gene.