HIV-1 encodes for the small basic protein Tat (86–101 residues) that drastically enhances the efficiency of viral transcription. The mechanism enabling Tat nuclear import is not yet clear, but studies using reporter proteins fused to the Tat basic domain indicate that Tat could reach the nucleus by passive diffusion. Tat also uses an unusual transcellular transport pathway. The first step of this pathway involves high-affinity binding of Tat to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2), a phospholipid that is concentrated in the inner leaflet of the plasma membrane and enables Tat recruitment at this level. Tat then crosses the plasma membrane to reach the outside medium. Although unconventional, Tat secretion by infected cells is highly active, and export is the major destination for HIV-1 Tat. Secreted Tat can bind to a variety of cell types using several different receptors. Most of them will allow Tat endocytosis. Upon internalization, low endosomal pH triggers a conformational change in Tat that results in membrane insertion. Later steps of Tat translocation to the target-cell cytosol are assisted by Hsp90, a general cytosolic chaperone. Cytosolic Tat can trigger various cell responses. Indeed, accumulating evidence suggests that extracellular Tat acts as a viral toxin that affects the biological activity of different cell types and has a key role in acquired immune-deficiency syndrome development. This review focuses on some of the recently identified molecular details underlying the unusual transcellular transport pathway used by Tat, such as the role of the single Trp in Tat for its membrane insertion and translocation.