Absorptive cells of the small intestine are highly polarized cells with distinct microvillus membrane (MVM) and basolateral plasma membrane domains. We compared membrane structure in the following preparations of rat small intestine commonly used for in vitro study of MVM function: epithelial sheets, isolated epithelial cells, and four different MVM vesicle preparations, using electron microscopy of thin sections and freeze fracture replicas. We also quantitated mean vesicle diameter of the four MVM preparations by quasielastic light scattering and determined their actin content. Epithelial sheets maintained their plasma membrane polarity as judged by intramembrane particle (IMP) distribution for at least 30 min after isolation. In contrast, the plasma membrane of isolated cells showed redistribution of IMPs, indicating considerable loss of polarity in the few minutes required for cell recovery. The P-face IMPs in MVM prepared by Ca++ precipitation were randomly distributed but became aggregated after exposure to potassium thiocyanate, which removed approximately 50% of core actin. The P-face IMPs in Mg++ precipitated MVM were aggregated whether or not core actin was depleted with potassium thiocyanate. The shape and size of MVM vesicles differed considerably with different preparative techniques. The extremely rapid loss of plasma membrane polarity of isolated intestinal epithelial cells and the striking structural heterogeneity of MVM vesicles prepared by commonly used techniques should be considered in the interpretation of functional studies with these preparations.