Glucagon-like peptide-2 (GLP-2) has recently been identified as a stimulator of intestinal epithelial growth, prompting the development of RIA and HPLC methodologies to study this peptide in more detail. A GLP-2-specific antiserum (UTTH-7) was developed that recognizes amino acids 25-30 of human and rat GLP-2-(1-33). UTTH-7 cross-reacts with N- and C-terminally modified forms of GLP-2, proglucagon, and the major proglucagon fragment. Analysis of rat ileal extracts demonstrated the presence of GLP-2-(1-33) as well as significant amounts of GLP-2-(3-33) (16 +/- 7% of total GLP-2). The level of total immunoreactive GLP-2 in plasma from fasted rats was 700 +/- 71 pg/ml, and this increased 3.6-fold (P < 0.001) in 24-h fed rats. HPLC analysis demonstrated the presence of both GLP-2-(1-33) and GLP-2-(3-33) in plasma from fasted rats, with increments in both peptides in plasma from fed rats. Immunoreactive GLP-2 increased in plasma from human subjects 2 h after a meal, rising from 851 +/- 230 to 1106 +/- 211 pg/ml (P < 0.05); 15 +/- 4% of this immunoreactivity was accounted for by the presence of intact GLP-2. HPLC showed the presence of both GLP-2-(1-33) and GLP-2-(3-33) in plasma from fed humans. Incubation of human GLP-2-(1-33) with the enzyme dipeptidylpeptidase IV resulted in liberation of GLP-2-(3-33), whereas replacement of Ala2 with Gly2 prevented this cleavage. Thus, while GLP-2-(1-33) is a major circulating and tissue form of GLP-2, GLP-2-(3-33) is a significant component ofimmunoreactive GLP-2 in both intestine and plasma.