Intestinal mucosal cells from the rat have been isolated by a new technique involving intravascular perfusion of an intestinal segment with collagenase. Detached cells were flushed from the intestinal lumen with a second perfusion circuit containing an oxygenated buffered solution with 1% bovine serum. Sequential collection of cells at intervals during the period of perfusion revealed that villus-tip cells are recovered first (after 15 min of collagenase perfusion), followed by midvillus (after 25 min) and lower villus cells (after 35 min). The isolated cells were judged intact and viable by the criteria of trypan blue dye exclusion, ultrastructural appearance, and metabolic activity. They were characterized as villus-tip, midvillus, and lower villus-crypt cells by their alkaline phosphatase and sucrase activity, glycoprotein formation, and [3H]thymidine incorporation. Microsomal monooxygenase activity was four to five times greater in villus-tip than in lower villus cells, whereas heme oxygenase exhibited a reverse gradient. The isolated cells synthesized heme and bilirubin under cell culture conditions.