Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of anti‐HIV‐1 Tat, exon 1 or exon 2 specific monoclonal antibodies have been used to construct single chain intracellular antibodies ‘intrabodies’ for expression in the cytoplasm of mammalian cells. These anti‐Tat single chain intrabodies (anti‐Tat sFvs) are additionally modified with a C‐terminal human C kappa domain to increase cytoplasmic stability and/or the C‐terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively. The anti‐Tat sFvs with specific binding activity against the N‐terminal activation domain of Tat, block Tat‐mediated transactivation of HIV‐1 LTR as well as intracellular trafficking of Tat in mammalian cells. As a result, the transformed lymphocytes expressing anti‐Tat sFvs are resistant to HIV‐1 infection. Thus, these studies demonstrate that stably expressed single chain intrabodies and their modified forms can effectively target molecules in the cytoplasm and nuclear compartments of eukaryotic cells. Furthermore, these studies suggest that anti‐Tat sFvs used either alone or in combination with other genetically based strategies may be useful for the gene therapy of HIV‐1 infection and AIDS.