Localization of cell membrane markers in dissociated frog urinary bladder epithelial cells was studied. The bladder cells began to alter in shape immediately after dissociation and became spherical within 15 min at room temperature. The apical marker, dialyzed iron (DI), was found on the entire surface of dissociated and transformed cells, whereas the basolateral marker, horseradish peroxidase-glutaral-dehyde (HRP-GLA) staining, first gathered at one pole and then became distributed over a larger area. Thus the apparent loss of polarity was not parallel with the true surface uniformity. When bladder cells were dissociated in the presence of cytochalasin B (CB) or 2,4-dinitrophenol (DNP), the transformation was suppressed and the cells maintained structural polarity. DI and HRP-GLA were mostly confined to the original region in CB-treated cells, but both moved to the other membrane region in DNP-treated cells. The results indicated that an aerobic adenosine triphosphate supply, as well as the tight junction, is involved in maintaining regional differentiation of the cell membrane.