Expression of the gene encoding glucagon was studied using a BK virus-induced glucagon-producing hamster islet cell line, InRl-G9 cells. Southern blot analysis of InRl-G9 DNA demonstrated that glucagon gene sequences are not amplified, yet appear to be hypomethylated compared to hamster liver or kidney DNA. Northern blot analysis of RNA from InRl- G9 cells detected a single glucagon mRNA species of 1300 basepairs. Phorbol esters and sodium butyrate, agents that increase glucagon gene transcription in RIN1056A cells, have no effect on glucagon mRNA levels in InRl-G9 cells. Posttranslational processing of proglucagon, as analyzed by gel filtration chromatography and RIA, resulted in the liberation of glucagon, glucagon-like peptide I, and glucagon-like peptide II, partially mimicking the processing of proglucagon in pancreas and intestine, yet differing from that previously observed in RIN1056A cells. Secretion of glucagon and the glucagon-like peptides was stimulated 3-fold after 1-h incubations with phorbol esters. These observations suggest that the determinants of glucagon gene expression and the posttranslational processing of proglucagon are highly cell specific and provide a new model for the study of proglucagon biosynthesis. (Endocrinology123: 1861–1867, 1988)