The locus control region of the beta-globin gene cluster has been used previously to direct erythroid expression of globin genes from retroviral vectors for the purpose of gene therapy. Short erythroid regulatory elements represent a potentially valuable alternative to the locus control region. Among them, the GATA-1 enhancer HS2 was used to replace the retroviral enhancer within the 3'-long terminal repeat (LTR) of the retroviral vector SFCM, converting it into an erythroid-specific regulatory element. In this work, we have functionally studied an additional GATA-1 enhancer, HS1. HS1 participates in the transcriptional autoregulation of GATA-1 through an essential GATA-binding site that is footprinted in vivo. In this work we identified within HS1 a new in vivo footprinted region, and we showed that this sequence indeed binds a nuclear protein in vitro. Addition of HS1 to HS2 within the LTR of SFCM significantly improves the expression of a reporter gene. The deletion of the newly identified footprinted sequence in the retroviral construct further increases expression up to a level almost equal to that of the wild type retroviral LTR, without loss of erythroid specificity, suggesting that this sequence may act as a negative regulatory element. An improved vector backbone, MDeltaN, allows even better expression from the new GATA cassette. These results suggest that substantial improvement of overall expression can be achieved by the combination of multiple changes in both regulatory elements and vectors.