A highly reproducible method is described for simultaneous preparation of the rabbit intestinal BBMV and BLMV. When BLMV were purified on continuous sorbitol gradient the final enrichment was over ten-fold for both Na+, K(+)-ATPase and K(+)-activated phosphatase. BBMV were separated using repeated precipitation with magnesium and EGTA. Marker enzymes of the brush border membrane also showed over ten-fold enrichment. There was no contamination with other cellular organelles. The characteristics of the preparations were checked by SDS gel electrophoresis, which showed distinct protein patterns for both membranes; monoclonal antibodies for villin reacted with the BBMV but not with BLMV, showing the absence of contamination of the BLMV fraction. The quality of the membranes, assessed by D-glucose transport, showed good functional properties for both BBMV and BLMV. Orientation of the vesicles was checked by permeabilizing with SDS buffered with BSA and showed that BLMV are oriented inside out and BBMV right side out. The model simulates normal transepithelial intestinal transport at the level of membrane vesicles and could be applied in transport studies in physiology and pharmacology.