Adenosine diphosphate-ribosylation factor 6 (ARF6), ARF6 mutants, and ARF1 were transiently expressed in Chinese hamster ovary cells, and the effects on receptor-mediated endocytosis were assessed. Overexpressed ARF6 localized to the cell periphery and led to a redistribution of transferrin receptors to the cell surface and a decrease in the rate of uptake of transferrin. Similar results were obtained when a mutant defective in guanosine triphosphate hydrolysis was expressed. Expression of a dominant negative mutant, ARF6(T27N), resulted in an intracellular distribution of transferrin receptors and an inhibition of transferrin recycling to the cell surface. In contrast, overexpression of ARF1 had little or no effect on these parameters of endocytosis.