Human immunodeficiency virus, type 1 (HIV-1) replication requires the interaction of Tat protein with the human cyclinT1 (hCyclinT1) subunit of the positive transcription elongation factor (P-TEFb) complex, which then cooperatively binds to transactivation response element (TAR) RNA to transactivate HIV transcription. In this report, a non-immune human single-chain antibody (sFv) phage display library was used to isolate anti-hCyclinT1 sFvs that could disrupt hCyclinT1-Tat interactions. The N-terminal 272 residues of hCyclinT1, including the entire cyclin domains and the Tat.TAR recognition motif (TRM), that fully support Tat transactivation was used for panning, and of the five unique anti-hCyclinT1 sFvs that were obtained, three bound to the cyclin box domains and two bound to TRM. All sFvs could be expressed as intrabodies at high levels in transiently transfected 293T and in stable Jurkat and SupT1 transfectants and could specifically co-immunoprecipitate co-expressed hCyclinT1 in 293T cells with varying efficacy without disrupting hCyclinT1-Cdk9 interactions. In addition, two sFv clones (3R6-1 and 2R6-21) that mapped to the cyclin box domains markedly inhibited Tat-mediated transactivation in several transiently transfected cell lines without inhibiting basal transcription or inducing apoptosis. When HIV-1 challenge studies were performed on stable 3R6-1-expressing Jurkat T cells, near complete inhibition of viral replication was obtained at a low challenge dose, and 74-88% inhibition to HIV-1 replication was achieved at a high infection dose in SupT1 cells. These results provide proof-in-principle that anti-hCyclinT1 intrabodies can be designed to block HIV-1 replication without causing cellular toxicity, and as a result, they may be useful agents for "intracellular immunization"-based gene therapy strategies for HIV-1 infection/AIDS.