Mutations producing beta-thalassemia reach individual gene frequencies greater than .01 in malarial-endemic regions because beta-thalassemia trait individuals have increased genetic fitness over that of normal individuals. Exon 3 of the beta-globin gene has been relatively spared as a site of common beta-thalassemia mutations. Frameshifts caused by the loss of a single nucleotide and nonsense mutations produce beta-thalassemia trait when they occur in exons 1 and 2. In contrast, they usually produce chronic hemolytic anemia when present in exon 3. Certain missense mutations in exon 3 produce unstable globins and thalassemia intermedia with hemolysis in heterozygotes. Here we report two new mutations in exon 3 of the beta-globin gene. One is a single nucleotide deletion in codon 109 in a 78-year-old Lithuanian with chronic hemolytic anemia and features of thalassemia. It leads to an abnormal globin (beta Manhattan) that is elongated to 156 amino acids. The second is a CAG-CGG missense mutation at codon 127 that causes a Gln----Pro substitution (beta Houston) and a thalassemia intermedia with hemolysis in three generations of a British-American family. Although the clinical phenotypes of these two patients differed little, differences in globin-synthetic ratios were significant, presumably reflecting differences in the ability of each abnormal beta-globin to form alpha beta dimers. The paucity of high-frequency exon 3 mutations and their worldwide distribution is likely attributable to their phenotypic severity and loss of increased genetic fitness vis-a-vis malaria.