Inhibition of beta-globin gene expression by antisense nucleic acids is a potentially powerful therapeutic strategy for sickle cell disease. To develop clinically relevant beta-globin antisense agents we created nine stable mouse erythroleukemia cell lines expressing unique anti-beta-globin RNA transcripts with different potentials for cross-hybridization with gamma-globin mRNA. We observed variable inhibition of beta-globin expression independent of the hybridization potential of the respective antisense beta-globin RNA transcript. Similarly, inhibition of gamma-globin expression by anti-beta transcripts varied widely in the nine stable cell lines. Three neighboring regions in the beta-globin gene with low RNA folding potentials conferred significantly stronger antisense effect toward beta-globin while sparing the homologous targets in gamma-globin. We have identified for the first time targets in the beta-globin gene for which the homologous regions in gamma-globin are relatively inaccessible to antisense attack. Our findings offer the prospect of using this approach to reduce the proportion of intracellular hemoglobin S. Gene therapy strategies which combine gamma-globin induction along with beta-globin inhibition using antisense vectors may yield more favorable anti-sickling effects longterm.