The functional properties of an epithelium are inextricably linked to its polarized structure. It has been difficult to study polarity at the level of the single cell, since most epithelial cells lose their polarity within minutes after dissociation. We have developed a preparation of native, dissociated, Ambystoma proximal tubule cells that maintain structural and functional polarity for a minimum of 7 days in suspension. We have used these cells to explore cell surface polarity in a single cell. Electron microscope cytochemical and immunocytochemical studies show that alkaline phosphatase is localized exclusively to the apical brush border, whereas the Na(+)-K(+)-ATPase is restricted to the basolateral membrane. Just as in the proximal tubule in situ, a sharp structural transition between the apical and basolateral membrane domains is retained. The ZO-1 protein found at the tight junction in situ is not present on the membrane of the dissociated cells, but rather it is distributed in the cytoplasm. The actin cytoskeleton also remains polarized in the single cells, and its distribution and organization appear to help maintain cell polarity. Electrophysiological measurements show that these cells remain viable at least as long as they remain structurally polarized. Patch-clamp recordings from both the apical and basolateral membranes show that the distribution of several ion channel proteins maintains functional polarity. We hypothesize that, despite loss of the intercellular "gate" and membrane-associated ZO-1, the socalled "fence" function of the tight junctional complex is retained in these dissociated proximal tubule cells. This preparation may serve as a useful single cell model with which to study epithelial polarity and membrane trafficking pathways.