The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to 3-galactosidase, horseradish peroxidase, RNase A, and domain Ill ofPseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-f-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages.Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.The potential for intracellular therapeutic use of proteins, peptides, and oligonucleotides has been limited by the impermeable nature of the cell membrane to these compounds. A wide variety of methods have been proposed for the delivery of proteins and other macromolecules into living cells for either experimental or therapeutic uses, including microinjection, scrape loading, electroporation, liposomes (1-7), bacterial toxins (8)(9)(10), and receptor-mediated endocytosis (11-16). Most of these methods are either inefficient or time-consuming, cause appreciable cell death, or result in uptake into intracellular vesicles without efficient cytoplasmic delivery. Several approaches (15-18) rely on binding of macromolecules to the cell surface, followed by internalization via the endocytic route. However, since proteins that have entered this pathway remain enclosed within lipid vesicles, they do not have access to the cell cytoplasm, most typically the target environment. It seems reasonable to assume that the escape from endocytic vesicles is the ratelimiting step in achieving true cellular delivery, yet many assays fail to measure this.Recently the Tat protein from human immunodeficiency virus 1 (HIV-1) was shown to enter cells when added exogenously (19,20). Tat protein can simply be added to medium at concentrations as low as 1 nM, and biological responses can be detected. Since the assay for this process was the transactivation of a transfected reporter gene, activity reflects those molecules that had been targeted to the nucleus, presumably after cytoplasmic delivery. The mechanism by which Tat traverses a membrane and the precise intracellular location of this event remain unclear. However, Tat binds efficiently to cells, with >107 sites per cell and then is internalized by an adsorptive endocytosis process (20). In characterizing the uptake process using iodinated Tat, Mann and Frankel (20) observed that only 3% of the Tat becameThe publication costs of this article were...