An anti-TAR RNA aptamer called R06, which binds tightly and specifically to the trans-activation responsive (TAR) element of the human immunodeficiency virus type 1 (HIV-1) through loop-loop interactions has been previously selected.(1) We used HIV-based retroviral vectors to express the R06 aptamer. Its synthesis was driven by the U16 snoRNA. We investigated the ability of this cassette to interfere with TAR-mediated transcription using HeLa P4 cells stably expressing the beta-galactosidase gene under the control of the HIV-1 5'LTR. We demonstrated that, upon HIV-1 infection, the beta-galactosidase activity was reduced in cells expressing the nucleolar U16-R06 transcript. The replication of HIV-1 in these cells was also reduced as shown by quantification of the HIV-1 protease gene 24 h post-infection. This effect was specific and related to the formation of R06 TAR complex as an aptamer with a mutated loop, which was no longer able to bind to TAR, did not show any effect. The nucleolus is likely a compartment of interest for targeting the TAR-protein complex responsible for the trans-activation of transcription of the HIV-1 genome.