A rapid method is described for the simultaneous preparation of both membranes of guinea-pig enterocytes, using simple differential centrifugation techniques. Basolateral membranes were purified on a Percoll gradient and the final yield of (Na+ + K+)-ATPase was 12.4% of the original activity with an enrichment factor of 12.6-fold. Purification of the brush-border fraction was achieved by a calcium-precipitation technique. The yield of alkaline phosphatase was 18.9% of the original activity with an enrichment of 17.5-fold. Both fractions could be obtained within 3 h of the original homogenization. The characteristics of the preparations were checked by negative-staining electron microscopy and by the determination of glucose uptake. The orientation of the basolateral vesicles was determined by measuring the Mg2+-ATPase and (Na+ + K+)-ATPase activities and the [3H]ouabain binding before and after treatment of the preparation with a mixture of deoxycholate and EDTA which transforms the vesicles into sheets. There was a 60% rise in (Na+ + K+)-ATPase activity and ouabain binding, but no change in Mg2+-ATPase activity. It was therefore concluded that 60% of the original preparation consisted of inside-out vesicles and 40% of membrane sheets.