Nondeletional gene mutations giving rise to alpha-thalassemia can be found at polymorphic frequency in Southeast Asia. Although the most common is hemoglobin Constant Spring (Hb CS), caused by a termination codon mutation (UAA --> CAA, Gln) in the alpha2-globin gene and resulting in reduced synthesis of the elongated alpha-globin variant, Hb Pakse (UAA --> UAU, Tyr) also has been observed at a significant prevalence. Western blot analysis of ghost membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from an individual with alpha-thal 1/Hb Pakse revealed the existence of a higher molecular weight globin of 18 kd consistent with an alpha(Pakse)-globin chain. The presence of alpha(Pakse)-globin on membranes of Hb Pakse-containing red blood cells affords an explanation for the severity of anemia observed in such patients. However, because the 2 Hb variants cannot be distinguished by current biochemical techniques, we developed a convenient single-tube polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) protocol for the simultaneous diagnosis of Hb CS and Hb Pakse by amplifying a short fragment covering the termination codon of the alpha2-globin gene. This PCR-SSCP method required no internal control coamplification or use of restriction enzymes and has the potential of identifying all the other possible termination codon mutations in a single reaction with only 1 pair of primers.