Background and Aims: The expression of the lymphocyte homing receptor and activation marker L-selectin is different in colon and small intestinal intraepithelial lymphocytes (IELs). In this study, the mechanism of this difference in L-selectin expression was investigated. Methods: L-selectin expression on lymphocytes was measured by flow cytometry. L-selectin messenger RNA (mRNA) was detected by reverse-transcription polymerase chain reaction. L-Selectin expression on peripheral lymphocytes was analyzed after incubation with cytokines, food and bacterial antigens, and homogenates of small and large bowel. Results: L-selectin was expressed by none of the small intestinal IELs but by 30% of those in the colon and by 60% of splenocytes. mRNA for L-selectin was detectable in isolated lymphocytes of all three sites. L-Selectin was downregulated in colon IELs during colitis and up- regulated in small intestinal IELs after in vitro culture for 48 hours. Incubation of splenocytes with small intestinal homogenates led to a rapid down-regulation of L-selectin (1% vs. 60% untreated). Preincubation with a metalloproteinase inhibitor prevented L-selectin loss. Conclusions: The mechanism of the differential expression of L-selectin in mouse small intestine and colon appears to be an increased functional activity of a metalloproteinase (sheddase) in the small intestine compared with the colon.