Small molecule inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been extremely successful but are associated with a myriad of undesirable effects and require lifelong daily dosing. In this study we explore an alternative approach, that of inducing intracellular immunity using designed, zinc finger-based transcription factors. Three transcriptional repression proteins were engineered to bind sites in the HIV-1 promoter that were expected to be both accessible in chromatin structure and highly conserved in sequence structure among the various HIV-1 subgroups. Transient transfection assays identified one factor, KRAB-HLTR3, as being able to achieve 100-fold repression of an HIV-1 promoter. Specificity of repression was demonstrated by the lack of repression of other promoters. This factor was further shown to repress the replication of several HIV-1 viral strains 10- to 100-fold in T-cell lines and primary human peripheral blood mononuclear cells. Repression was observed for at least 18 days with no significant cytotoxicity. Stable T-cell lines expressing the factor also do not show obvious signs of cytotoxicity. These characteristics present KRAB-HLTR3 as an attractive candidate for development in an intracellular immunization strategy for anti-HIV-1 therapy.