The HIV-1 trans-activation responsive element (TAR) RNA 59-residue stem−loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibition of these interactions blocks full-length HIV transcription and hence replication. We have found that three types of 12-residue oligonucleotide analogues, namely, a 2‘-O-methyl oligoribonucleotide (OMe), a chimeric oligonucleotide containing 7×OMe and 5×5-methyl C locked nucleic acid (LNA) residues, and a peptide nucleic acid (PNA), inhibit Tat-dependent in vitro transcription in HeLa cell nuclear extract equally efficiently (50% inhibition at 100−200 nM) and sequence specifically. The results are correlated with surprisingly similar binding strengths to a model 39-residue TAR under transcription conditions. A 12-mer containing 11 contiguous LNA residues was less effective in both Tat-dependent transcription inhibition and TAR 39 binding. Anti-TAR 3‘-carboxyfluorescein- (FAM-) labeled OMe and OMe/LNA chimeric 12-mers were also efficient Tat-dependent in vitro transcription inhibitors as were 3‘-FAM-labeled OMe oligonucleotides containing some phosphorothioate (PS) linkages. By use of a HeLa cell line containing stably integrated plasmids expressing firefly luciferase under HIV-LTR/Tat dependence as well as a Renilla luciferase constitutive control, we showed submicromolar, selective, dose-dependent, and sequence-dependent intracellular inhibition of Tat-TAR trans activation by the anti-TAR 3‘-FAM 12-residue 7×OMe/5×LNA oligonucleotide when delivered by cationic lipid. No intracellular activity was observed for the corresponding anti-TAR 3‘-FAM OMe 12-mer. An alternating PS-containing 3‘-FAM OMe 12-mer oligonucleotide exhibited partial inhibition of trans-activation activity, but this was correlated with a similar effect on control gene expression, suggesting nonspecific inhibition.