Complex interrelationships exist between the four pancreatic islet cell types and their respective secretory products, insulin, glucagon, somatostatin, and pancreatic polypeptide. These hormones are known to interact with the different islet cells and modulate their functions. Insulin inhibits glucagon secretion from the A cell both in vivo and in vitro and, in states of insulin deficiency, high glucagon levels are observed that are normalized by insulin replacement. To determine if insulin also regulates glucagon biosynthesis, we studied its effects on glucagon gene expression. Our studies indicate that insulin, in a dose-dependent fashion decreases steady-state glucagon mRNA levels in a clonal hamster islet cell line, In-R1-G9; this decrease is secondary to an inhibition of glucagon gene transcription as assessed by transcriptional run-on assays and does not involve detectable changes in mRNA stability. Inhibition of glucagon gene transcription is accompanied by corresponding decreases in glucagon immunoreactivity in both cell extracts and medium. We conclude that insulin may not only regulate glucagon secretion but also glucagon gene expression.