A marked tissue-specific increase in erythrocyte adenosine deaminase (ADA) activity is associated with an autosomal dominantly inherited hemolytic anemia. We investigated the molecular basis of ADA overproduction by studying reticulocyte ADA mRNA from affected individuals. Analysis of proband reticulocyte ADA cDNA clones revealed normal sequence. RNase mapping demonstrated that the amount of ADA mRNA in affected reticulocytes was greater than the amount in normal B lymphoblasts, whereas ADA mRNA was undetectable in normal reticulocytes. The 5′- and 3′-untranslated regions of reticulocyte and B-lymphoblast ADA mRNAs from affected individuals were structurally indistinguishable from those of normal B lymphoblasts. Northern blot analysis performed under stringent hybridization and washing conditions confirmed a markedly increased amount of reticulocyte ADA mRNA in affected individuals as compared with controls. We conclude that the RBC-specific overexpression of ADA in this disorder occurs at the mRNA level.