Glucagon-like peptide 2 (GLP-2) appears to regulate growth of the small bowel epithelium. The importance of dose, vehicle, and route of administration for GLP-2 bioactivity was examined in vivo. Mouse small bowel weight increased following subcutaneous injection of GLP-2, 0.25 to 5.0 microg twice daily, with an increment in jejunal crypt plus villus height detected with only 250 ng GLP-2 twice daily for 14 days (1.35-fold greater than control, P < 0.05). Small bowel weight was increased by 6 days of GLP-2 administration (1.4-fold greater than control, P < 0.05) and further increased after up to 14 days of GLP-2 treatment (P < 0.001, GLP-2 vs. control). GLP-2 increased small bowel weight in either 12% gelatin or phosphate-buffered saline (PBS) formulations (P < 0.01 vs. P < 0.05, gelatin vs. PBS, respectively), and GLP-2 was intestinotrophic following either intramuscular, intraperitoneal, or subcutaneous administration (P < 0.05 for im and ip, P < 0.001 for sc, GLP-2 vs. control). GLP-2 also induced bowel growth when administered daily or every other day. These results define important biological parameters for the activity of GLP-2 in vivo.