We sought to investigate the usefulness of the adeno-associated virus 2 (AAV)-based vectors to suppress the excess production of the human α-globin gene product towards developing a treatment modality for β-thalassemia since accumulation of free α-globin reduces the lifespan of red blood cells in these patients. We constructed recombinant AAV virions containing the human α-globin gene sequences in antisense orientation driven by the herpesvirus thymidine kinase (TK) promoter, the SV40 early gene promoter, and the human α-globin gene promoter, respectively, as well as a bacterial gene for resistance to neomycin (neo(R)) as a selectable marker. These recombinant virions were used to infect a human erythroleukemia cell line (K562) that expresses high levels of α-globin mRNA. Clonal populations of neo(R) cells were obtained after selection with the drug G418, a neomycin analogue. Total genomic DNA samples isolated from these cells were analyzed on Southern blots to document stable integration of the transduced neo and α-globin genes. Total cellular RNA samples isolated from mock-infected and recombinant virus- infected cultures were also analyzed by Northern blots. Whereas the TK promoter-driven antisense α-globin sequences showed no inhibition of expression of the endogenous α-globin gene, the SV40 promoter and the α- globin gene promoter-driven antisense α-globin sequences suppressed the expression of this constitutively over-expressed gene by approximately 29 and 91%, respectively, at the transcriptional level. These studies suggest the feasibility of utilizing the AAV-based antisense gene transfer approach in the potential treatment of β-thalassemia.