To define the biological significance of the initial cleavage at the proglucagon (PG) interdomain site, K70-R71, we have created two interdomain mutants, K70Q-R71Q and R71A. Co-transfection studies in GH4C1 cells show significant amounts of glucagon production by PC2 along with some glicentin, GRPP (glicentin-related polypeptide)-glucagon and oxyntomodulin from wild-type PG. In contrast, a larger peptide, PG 33-158, and low amounts of GRPP-glucagon are predominantly generated from interdomain mutants. HPLC analysis shows a 5-fold increase in glucagon production by PC2 from wild-type PG and a corresponding 4-fold lower accumulation and secretion of unprocessed precursor relative to interdomain mutants. PC2 generates significant levels of glucagon from a glicentin (PG 1-69) expression plasmid whereas PC1/3 produces only modest amounts of oxyntomodulin. Employing a MPGF (major proglucagon fragment) (PG 72-158) expression plasmid, we show that PC1/3 predominantly generates GLP-1 whereas PC2 produces only N terminally extended GLP-1. Surprisingly, production of GLP-1 and GLP-2 by PC1/3 from interdomain mutants compared with wild-type PG is not significantly impaired. In addition to PC2 and PC1/3, PC5/6A and furin are also able to cleave the sites, K70-R71 and R107-X-R-R110 in PG. We show a much greater ability of furin to cleave the monobasic site, R77, than at the dibasic site, R124-R125, which is also weakly processed by PC5/6A indicating overlapping specificities of these two convertases mainly with PC1/3. We propose here a trimer-like model of the spatial organization of the hormonal sequences within the proglucagon molecule in which the accessibility to prohormone convertase action of most cleavage sites is restricted with the exception of the interdomain site, K70, R71, which is maximally accessible.