Glucagon-like peptide 2 (GLP-2) is a potent intestine-specific growth factor. To determine whether GLP-2 has a direct effect on cellular proliferation, we examined the effects of GLP-2 on three different intestinal epithelial cell lines: Caco-2, T84, and IEC-6. Proliferation was assessed by measuring [3H]thymidine incorporation into cellular DNA. The expression of the cell cycle regulators cyclins D1, E, and A was determined by western blotting. GLP-2 stimulated [3H]thymidine incorporation in Caco-2 and T84 cells but not in IEC-6 cells. In Caco-2 cells, increased [3H]thymidine incorporation was detectable by day 1 and was maximal at day 5 following initiation of daily GLP-2 administration. Increased [3H]thymidine incorporation persisted for three days following treatment with a single dose of GLP-2. GLP-2 administration resulted in the induction of cyclins D1, E, and A in Caco-2 cells. These results suggest that GLP-2 acts directly on human intestinal epithelial cells to stimulate their proliferation.