Seven rabbits were immunized with a synthetic C-terminal glucagon fragment [15-29] conjugated with bovine serum albumin by means of glutaraldehyde. Antisera for glucagon were produced in all the animals after six injections of the conjugate. One of them revealed a higher titer antiserum (G 42), which did not cross react with gut glucagon-like immunoreactive material, secretin, insulin, gastric inhibitory polypeptide or vasoactive intestinal peptide. From the results of inhibition of 125I-glucagon in binding with the antiserum by various glucagon-related fragments the immunogenic determinant of the antiserum was proved to be in the C-terminal residue of the glucagon molecule, although peptide (17-29) or [21-29] reacted weakly with the antiserum. The plasma glucagon levels measured by antiserum G 42 during an arginine test in five normal subjects were superposed on those obtained by other antiserum (G21), specific for pancreatic glucagon. Furthermore, a comparable standard curve for glucagon was obtained using antiserum G42, when a labelled p-hydroxyphenylacetylated glucagon fragment [15-291 was employed as a tracer. The present study clearly demonstrated that the C-terminal glucagon fragment could yield a specific antiserum for pancreatic glucagon, supporting the proposal that the C-terminal fragment of glucagon is responsible for such specific antisera. Furthermore, it is concluded that immunoassay for glucagon could be performed using the labelled glucagon fragment as a tracer. 1 A part of this paper was presented at the Annual Meeting of the Eastern Branch of Japan Endocrine Society on October 8, 1977. The study was supported in part by a Scientific Research Grant of the Ministry of Education of Japan (no. 257169).