Centaurin beta 1 and Centaurin beta 2 are recruited to platelet-derived growth factor-induced dorsal membrane ruffles in NIH 3T3 mouse fibroblasts, and overexpression inhibited ruffle formation. In vitro, Centaurin beta 1 and Centaurin beta 2 preferred ARF6 as substrate rather than ARF1 or ARF5, and the GAP activity of both Centaurins was dependent upon phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The isolated PH domain of mouse Centaurin beta 2 exhibits moderate affinity for PtdIns(3,5)P2, but it does not bind any other phosphoinositides tested, including PtdIns(4,5)P2. Mutation of a highly conserved arginine in both Centaurins result in lack of ARF-GAP activity. Overexpression in HeLa cells of either Centaurin blocks the formation of ARF6-dependent protrusions. Both Centaurins are also recruited to peripheral, tubular membranes, where activation of ARF6 occurs and allows membrane recycling back to the plasma membrane.