The in-vitro synthesis of hemoglobin (Hb) chains was studied among 60 Hb S heterozygotes (AS) having different quantities of Hb S, including five with an associated alpha-chain heterozygosity (ASAG). Hematologic values and hemoglobin composition were studied in these cases and in 15 other ASAG heterozygotes. The percentages of Hb S (which fell between 27% and 42%) and the mean corpuscular volume values correlated directly with the alpha/non-alpha values, confirming previous suggestions (Huisman, Hemoglobin 1:349, 1977) that the concomitant occurrence of an alpha-thalassemia-2 heterozygosity (alpha alpha(0)/alpha alpha; beta/beta(S)) or homozygosity (alpha(0) alpha/alpha(0) alpha; beta/beta(S)) resulted in intermediate or lower levels of Hb S compared with Hb S heterozygotes having four active alpha-chain genes (alpha alpha/alpha alpha; beta/beta(S)). Among ASAG heterozygotes, the occurrence of low (about 25%), intermediate (about 33%), or high (about 45%) proportions of an alpha-chain variant resulting from a variability in the number of active alpha-chain genes due to alpha-thal-2 coincided with high (39%), intermediate (34%), or low (28%) levels of Hb S, respectively. However, the overlap of biosynthetic data between Hb S heterozygotes with four, three, or two active alpha-chain genes prevents a reliable diagnosis in individual cases.