We have been studying the cellular function of the ADP-ribosylation factor (ARF) 6 protein in mammalian cells. ARF6 is a member of the ARF family of Ras-related GTP-binding proteins. Transient transfection of cells with epitope-tagged, wild type ARF6 and mutants of ARF6, suggests that ARF6 is distributed within the cell according to its nucleotide status; it localizes to the plasma membrane (PM) in its GTP-bound state and to a unique, tubular, endosomal membrane compartment in its GDP-bound state. Whereas, the wild type protein in transfected cells is equally distributed along the PM and associated with the tubular endosomes, we have identified two pharmacologic treatments which shift this distribution. Treatment of these cells with the G protein activator aluminum fluoride causes a shift of the protein to the PM and results in the formation of cell protrusions at the PM, enriched in actin filaments as well as many actin associated proteins. These peripheral membrane protrusions resemble those observed in cells expressing the constitutively active (GTP-bound) ARF6 mutant. In contrast, treatment of cells with inhibitors of actin polymerization, such as cytochalasin D, causes the ARF6 wild type protein to shift its distribution off the PM to the internal membrane compartment. This tubular endosomal compartment is not transferrin receptor-positive and thus is not the well-studied receptor-mediated endocytosis pathway, and yet, we show that PM proteins move into and out of this compartment. Thus, it appears to represent a novel recycling compartment through which PM proteins cycle. The association of ARF6 with this PM recycling compartment suggests that ARF6, through its GTP cycle, may regulate membrane movement and actin polymerization at the cell periphery, possibly influencing cell shape and migration. With ARF-6 specific anti-peptide antibodies, we have found ARF6 expressed in all cell and tissue types examined. With this antibody, the endogenous ARF6 protein has been localized to the peripheral PM and internal structures, similar to that observed in the transfected cells.