We have investigated the role of intracellular HIV-1 Tat on CXCR4 expression on T cells. We found that stable or doxycycline-regulated expression of HIV-1 Tat on Jurkat T cells results in lower cell surface expression of CXCR4, but not of other chemokine receptors. This effect was not due to an alteration in CXCR4 transcription, and total CXCR4 levels remained unaltered. Rather, when cells were treated with CXCL12/Stromal Cell-Derived Factor 1, a faster downmodulation of CXCR4 was observed although resurfacing was unaffected. Similar effect was seen in peripheral human T cells transiently transfected with Tat. At the molecular level Tat did not alter cellular levels of G-coupled receptor kinases 2 and 6 and beta-arrestin, proteins involved in CXCR4 downregulation. Neither Tat significantly affected phosphatidylinositol 3-kinase activation in response to CXCL12. Interestingly, in Jurkat cell clones stably expressing both Protein kinase (PK)-Czeta and HIV-1 Tat, CXCL12 induced a faster CXCR4 internalization than in cells only expressing HIV-1 Tat. In contrast in Jurkat cell stably expressing a dominant negative PKCzeta, Tat enhancement of CXCR4 internalization was abrogated. Thus, our results show a new function of HIV-1 Tat, its ability to regulate CXCR4 expression via PKCzeta. The significance of those results is discussed.