Tat protein from human immunodeficiency virus can deliver biologically active proteins in vivo and is of considerable interest for protein therapeutics. The mechanism responsible for Tat-fusion protein internalization is still poorly understood and controversial. The punctuate distribution, timing, and temperature sensitivity observed in our experiments with Tat-fusion proteins are consistent with endocytosis. After a few hours, Tat-fusion proteins accumulated around the nucleus without any significant visible nuclear targeting. Using a Cre/Lox based functional assay, lysosomotropic agents known to disrupt endosome integrity, increased by up to 23-fold the nuclear delivery of functional Tat-Cre recombinase without increasing cell uptake in a similar fashion. This shows that endosome disruption can significantly increase Tat-fusion protein access to the cytosol and nucleus. In addition, we found that internalized Tat-fusion proteins persisted several hours and that inhibitors of lysosome acidification did not increase functional nuclear delivery of Tat-Cre. This suggests that Tat-fusion proteins enter via the endosomal pathway, circumvent lysosomal degradation, and are then sequestered in the periphery of the nucleus. Most importantly, our work indicates that an inadequate intracellular trafficking is the main factor limiting the efficiency of protein cargo delivery using Tat. (C) 2004 Elsevier Inc. All rights reserved.