Enteroglucagon (II) was isolated from extracts of pig ileum mucosa by repeated gel filtrations, and its immunochemical and chromatographic characteristics were compared with those of a synthetic peptide corresponding to the 33-69 sequence of pig glicentin, before and after digestion with trypsin or trypsin followed by carboxypeptidase B, by using five region-specific assays covering most of the glicentin sequence. Enteroglucagon (II) and the synthetic peptide behave identically under three different conditions of chromatography as determined with all five assays (including a highly specific radioreceptor assay), and gave rise to similar fragments after enzyme digestion. It was therefore concluded that enteroglucagon (II) and the 33-69 sequence of glicentin are most probably identical.