This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested, HUT 78 and Raji. These results are consistent with previous observations that upstream promoters exert an inhibitory effect on promoters placed downstream and bear out the predictions used in the design of DC vectors. The use of DC vectors may contribute to the solution of the problems encountered in expressing retrovirally transduced genes in cultured cells and, in particular, when introduced into the live animal.