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The extent to which a donor membrane will be retrieved, or if it is retrieved at all after it fuses with an acceptor membrane, is usually difficult to determine. We have studied the dynamics of membrane retrieval in the phagosome system of Paramecium multimicronucleatum using six monoclonal antibody markers. Our previous freeze-fracture and transmission electron microscopic studies have indicated that extensive changes take place in the membrane of the young phagosome as it progresses through its cycle. Using immunofluorescence and immunoelectron microscopy to determine the times of entry and exit of these individual antigens into the digestive vacuole system, we showed that two hydrophilic antigens, one located on the cytosolic and one on the lumenal side of the discoidal membrane (phagosome membrane precursor), were completely retrieved from the phagosome by tubulation within the first three minutes. At the same time that this membrane was retrieved, membrane from a second population of vesicles, the acidosomes, fused with the phagosome to produce the phagoacidosome. On the basis of immunogold localization on cryosections of a total of six antigens, the two specific for phagosome/discoidal vesicle membrane as well as four specific for the acidosome/phagoacidosome membrane, this replacement is total. We also showed that in the presence of the actin-active drug cytochalasin B, this replacement was essentially prevented. However, when vacuole acidification was neutralized by ammonium chloride, this replacement process continued unaffected after a lag. Consequently, acidification, per se, is not required to trigger the replacement of the phagosome membrane. We conclude, on the basis of these studies as well as our previous freeze-fracture studies that during phagoacidosome formation most of the acceptor membrane is retrieved and is replaced by the donor membrane. This shows that at least one cell type possesses the mechanisms needed to substantially replace the membrane of a phagosomal compartment when radical and rapid changes are needed to modulate the digestive and absorptive processes. more...