Actin filament organization is essential for endocytosis in yeast. In contrast, the actin-depolymerizing agent cytochalasin D has yielded ambiguous results as to a role for actin in receptor-mediated endocytosis in mammalian cells. We have therefore re-examined this issue using highly specific reagents known to sequester actin monomers. Two of these reagents, thymosin beta4 and DNase I, potently inhibited the sequestration of transferrin receptors into coated pits as measured in a cell-free system using perforated A431 cells. At low concentrations, thymosin beta4 but not DNase I was stimulatory. Importantly, the effects of both reagents were specifically neutralized by the addition of actin monomers. A role for the actin cytoskeleton was also detected in intact cells where latrunculin A, a drug that sequesters actin monomers, inhibited receptor-mediated endocytosis. Biochemical and morphological analyses suggest that these reagents inhibit later events in coated vesicle budding. These results provide new evidence that the actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells.