The human alpha-globin genes are duplicated and encode identical polypeptides. Recently we detected in cloned genomic DNAs characteristic sequence differences between the 3' untranslated regions of the 5' (alpha 2) and 3' (alpha 1) genes, not previously recognized by direct analysis of mRNA and cDNA transcripts. Based on these untranslated region differences, we have now used S1 nuclease mapping of RNA to detect and quantitate the two predicted alpha-mRNA species. With this assay we have examined the relative expression of the alpha-globin genes during normal development and in alpha-thalassemia syndromes. In normal adult reticulocytes, alpha 2 RNA is slightly more abundant than the alpha 1 species (ratio 60:40). This relative abundance of the alpha RNAs was consistently observed in fetal blood and liver RNA samples from 10 weeks of gestation to birth. In both deletion and nondeletion forms of alpha thalassemia, only the alpha 1 RNA and establish the normal pattern of relative alpha-gene expression during development independent of protein variants. RNA analysis also permits for the first time identification of the mutant genes in nondeletion forms of thalassemia.