The Tat protein from human immunodeficiency virus 1 (HIV-1) can efficiently enter cells when added exogenously. This study demonstrates that Tat can deliver heterologous proteins into cells by chemically cross-linking Tat peptides to various proteins such as β-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE). The Tat-chimeras were effective in delivering these proteins to various cell types, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-β-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, and low-to-moderate levels in lung and skeletal muscle. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages. 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. Various methods have been proposed for the delivery of proteins and other macromolecules into living cells, including microinjection, scrape loading, electroporation, liposomes, bacterial toxins, and receptor-mediated endocytosis. However, most of these methods are inefficient or time-consuming, cause appreciable cell death, or result in uptake into intracellular vesicles without efficient cytoplasmic delivery. The Tat protein can be used to deliver heterologous macromolecules into cells. This study shows that Tat peptides conjugated to heterologous proteins can confer cellular delivery to these "cargo" proteins. Delivery is independent of cell type, has been successfully applied to four different cargo proteins, and has been shown unambiguously to mediate cytoplasmic delivery. Preliminary in vivo experiments confirm that this system may allow the therapeutic use of proteins/peptides with intracellular targets. The study also shows that Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells. The Tat protein can enter cells when added exogenously and can be used to deliver heterologous proteins into cells. The Tat protein binds efficiently to cells and is internalized by an adsorptive endocytosis process. The study also shows that the basic RNA binding domain of Tat is involved in mediating cell surface binding of Tat. The study also shows that the use of chloroquine can reduce the degradation of the Tat protein and enhance its transactivation activity. The study also shows that the Tat protein can be used to deliver various proteins into cells, including β-galactosidase, horseradish peroxidase, RNase A, and domain III of PE. The study also shows that the Tat protein can be used to deliver these proteins into various tissuesThe Tat protein from human immunodeficiency virus 1 (HIV-1) can efficiently enter cells when added exogenously. This study demonstrates that Tat can deliver heterologous proteins into cells by chemically cross-linking Tat peptides to various proteins such as β-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE). The Tat-chimeras were effective in delivering these proteins to various cell types, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-β-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, and low-to-moderate levels in lung and skeletal muscle. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages. 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. Various methods have been proposed for the delivery of proteins and other macromolecules into living cells, including microinjection, scrape loading, electroporation, liposomes, bacterial toxins, and receptor-mediated endocytosis. However, most of these methods are inefficient or time-consuming, cause appreciable cell death, or result in uptake into intracellular vesicles without efficient cytoplasmic delivery. The Tat protein can be used to deliver heterologous macromolecules into cells. This study shows that Tat peptides conjugated to heterologous proteins can confer cellular delivery to these "cargo" proteins. Delivery is independent of cell type, has been successfully applied to four different cargo proteins, and has been shown unambiguously to mediate cytoplasmic delivery. Preliminary in vivo experiments confirm that this system may allow the therapeutic use of proteins/peptides with intracellular targets. The study also shows that Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells. The Tat protein can enter cells when added exogenously and can be used to deliver heterologous proteins into cells. The Tat protein binds efficiently to cells and is internalized by an adsorptive endocytosis process. The study also shows that the basic RNA binding domain of Tat is involved in mediating cell surface binding of Tat. The study also shows that the use of chloroquine can reduce the degradation of the Tat protein and enhance its transactivation activity. The study also shows that the Tat protein can be used to deliver various proteins into cells, including β-galactosidase, horseradish peroxidase, RNase A, and domain III of PE. The study also shows that the Tat protein can be used to deliver these proteins into various tissues