This study was done to establish and validate a single-pass perfusion method for measuring the absorption of water and electrolytes by the mouse small intestine. The method was then used to study intestinal absorption in mice whose villin gene had been invalidated (v/). The single-pass perfusion of the jejunum measures the absorption of water, Cl, Na⁺, K⁺, HCO, and glucose in anesthetized wild-type and v/ mice in vivo. We measured absorption under basal and stimulated conditions (carbachol, vasoactive intestinal polypeptide, intralumen PGE₂). Basal absorption and stimulated secretions were similar to those previously obtained in rats. There was no difference between wild-type and v/ mice in animals with mixed genetic background or in pure C57BL6 mice. We conclude that this in vivo perfusion method is suitable for studying the absorption/secretion of electrolytes in the mouse intestine and that a lack of villin does not significantly alter basal and secretagogue-stimulated electrolyte movements across the epithelium of the mouse jejunum in vivo.