Polymorphism for a Hpa I restriction endonuclease site associated with about 60% of beta S genes in American Blacks allows exact prenatal diagnosis of sickle cell anemia by amniocentesis in 36% of couples at risk. In three families in whom exact diagnosis by Hpa I sites was impossible, we found analysis for the presence of polymorphic HindIII sites in the G gamma and A gamma intervening sequences would allow an exact prenatal diagnosis of sickle cell status in all three. In one of these families, the presence of an A gamma HindIII site in amniocyte DNA confirmed the diagnosis (sickle cell trait) made by synthetic studies using fetal erythrocytes obtained at fetoscopy. Studies of other Black families and individuals provide evidence for linkage disequilibrium in the G gamma-A gamma-delta-beta gene complex involving the four sites, G gamma HindIII, A gamma HindIII, beta S, and Hpa I, which span 33 kilobases (kb). Ten of 14 chromosomes bearing a beta S gene in a 7.6-kb Hpa I fragment contained a G gamma but not an A gamma HindIII site, whereas 16 of 16 chromosomes bearing a beta S gene in a 13-kb Hpa I fragment lacked both the G gamma and A gamma HindIII sites. Two-thirds of beta A-bearing chromosomes lacked both G gamma and A gamma sites, whereas one-third contained either the G gamma or both G gamma and A gamma sites. These data demonstrate that combined analysis of both Hpa I and HindIII polymorphisms and verification of their linkage phase should increase the fraction of couples for whom amniocentesis can provide an exact diagnosis of sickle cell status from 36% to greater than 80%.