We constructed a full open reading frame (ORF) for the rat hepatic glucagon receptor using two non functional pCDM8 clones of a putative glucagon receptor. Clone I was complete but contained, in addition, two small introns interrupting the frame, while clone II lacked 104 bp at the 5' end. We isolated Hind III/Dra III and BamH I/Not I fragments from clone I and a Dra III/BamH I fragment from clone II then ligated the three fragments with pBluescript SK(+) digested with Hind III/Not I. Following this 4 fragment ligation procedure, we obtained one correct insert in a clone we subcloned in the Hind III/Not I sites of pCDM8. This plasmid was used to transiently transfect COSGs1 cells with the lipofectin method. In COSGs1 membranes transfected with this correct plasmid (but not with a truncated plasmid) [125I]iodoglucagon binding was selectively displaced with glucagon (IC50 = 4 nM) and adenylate cyclase was stimulated with glucagon (K(act) = 10 nM) while related peptides were inefficient at 1 microM. This demonstrated that the receptor previously described is indeed the rat hepatic glucagon receptor.