Abstract In this report we demonstrate that approximately 1.1 kb of the rat glucagon gene promoter upstream of the transcriptional start site specifically directs the transcription of the reporter gene chloramphenicol acetyl transferase (CAT) (p[-1.1]GLU-CAT) in insulinoma β-TC1 cells. On the contrary, the 350 bp closest to the transcription start site (p[-0.35]GLU-CAT) are ineffective in β-TC1 cells. Both constructs are transcriptionally active in InR1-G9 glucagonoma cells. While protein kinase A and protein kinase C activators, acting through independent pathways, strongly increase both the transcription of p[-1.1]GLU-CAT and the accumulation of glucagon transcript in β-TC1 cells, they are weaker activators in InR1-G9 cells. Our experiments suggest that some positive transcription control elements, necessary for the glucagon gene transcription in insulinoma β-TC1 cells, are localized in the -350/-1100 region of the glucagon gene. Furthermore, our data indicate that glucagon gene transcription can be strongly activated through the protein kinase A pathway in some specific cellular contexts.