We have developed an in vitro system for studying membrane transport during receptor-mediated endocytosis. Using nitrocellulose disruption to permeabilize selectively the apical domain of filter-grown MDCK cells, the recycling of receptor-bound transferrin (Tfn) from an intracellular pool was reconstituted in vitro with a rate and efficiency similar to that of intact cells. Tfn and Tfn receptor recycling from endosomes back to the cell surface was dependent on added ATP and cytosol-derived proteins. Thus, incubation of intact cells under conditions of ATP depletion resulted in the clearance of Tfn receptors from the basolateral membrane, this was reversible upon removal of the energy poisons. Reappearance of previously internalized receptors could also be obtained in disrupted cells but required the addition of both ATP and cytosol to the assay mixture. Similarly, when intact cells were allowed to internalize labeled Tfn prior to disruption, efficient and rapid release of ligand back into the medium was markedly stimulated by ATP and cytosol. Recycling was judged to be both selective and vectorial since only the expected small fraction of a previously internalized horseradish peroxidase was released after addition of ATP and cytosol, and release was primarily into the basal medium. While the cytosol contributed one or more protein factors, none was sensitive to N-ethylmaleimide. Alkylation of the disrupted cells, however, did inactivate recycling.