Targeting and recruitment of the plasma membrane (PM) clathrin-coated vesicle adaptor complexes has been studied using an in vitro system based on permeabilized acceptor cells and donor cytosol. Through the use of species- and/or tissue-specific antibodies, only newly recruited exogenous PM adaptors are visualized. Targeting of PM adaptors can be switched from the plasma membrane to a perinuclear compartment by GTP gamma S or excess calcium. Prior treatment with brefeldin A prevents GTP gamma S-induced mistargeting. Double-labeling immunofluorescence and immunogold EM indicate that the perinuclear PM adaptor binding compartment is late endosomal. We propose that receptors for PM adaptors cycle between the plasma membrane and an endosomal storage compartment. Normally the receptors would be switched on only at the plasma membrane, but both GTP gamma S and calcium are capable of reversing this switch. Intracellular sequestration of PM adaptor receptors may provide the cell with a mechanism for up-regulating endocytosis following a burst of exocytosis.