Intracellularly expressed antibodies, referred to as "intrabodies" can be designed to bind and inactivate target molecules inside cells. In our previous study, mammalian cells were transduced to produce an anti-gp120 single-chain intrabody sFv105 to inactivate human immunodeficiency virus type-1 (HIV-1) infection. Here, an inducible expression vector was constructed in which the sFv105 intrabody, which reacts with the CD4-binding site of HIV-1 gp120, is under the control of the HIV-1 long terminal repeat (LTR)/promoter. The sFv105 intrabody is inducibly expressed after HIV-1 infection or in the presence of Tat protein and is retained intracellularly. A human CD4+ lymphocyte line transformed with the expression vector exhibits resistance to the virus-mediated syncytium formation and a decreased ability to support HIV-1 production. Surface gp120 expression is markedly reduced and surface CD4 is restored to normal following HIV-1 infection in the transformed lymphocytes. Cell-surface phenotype, replication rate, morphology, and response to mitogenic stimulation of the transformed cells are also normal. Thus, intrabodies are a new class of active molecules that may be useful for the gene therapy of acquired immunodeficiency virus (AIDS) and other diseases.