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PDX-1 plays a central role in regulating insulin gene transcription and differentiation of insulin-producing cells. It was previously reported that, due to its own Antennapedia-like protein transduction domain (PTD), exogenous PDX-1 protein can permeate cells and induces insulin gene expression in pancreatic ducts, thought to be islet progenitor cells. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells without requiring gene transfer technology. Here it is shown that after an initial ionic cell–surface interaction, PDX-1 proteins are rapidly internalized by lipid raft-dependent macropinocytosis. HeLa cells were treated with both FITC-conjugated PDX-1 PTD and FM 4–64, a general fluorescent marker of endocytosis. A punctate cytoplasmic distribution of PDX-1 PTD, which colocalized with FM 4–64, was observed in treated cells. Because expression of dominant-negative dynamin-1 did not block PDX-1 PTD uptake, PDX-1 protein transduction is independent on phagocytosis and clathrin- or caveolar-mediated endocytosis. Cells were pretreated with amiloride, a specific inhibitor of the Na+/H+ exchange required for macropinocytosis, or cytochalasin D, an F-actin elongation inhibitor. Treatment of cells with both macropinosome inhibitors resulted in the reduction in PDX-1 PTD transduction into vesicles, suggesting that PDX-1 PTD-mediated cellular entry occurs by lipid raft-mediated macropinocytosis. Taken together, these observations provide the mechanism of PDX-1 protein transduction and suggest that the protein transduction system could work for experimental and therapeutic strategies.