Proinsulin is converted to insulin by the two endoproteases PC2 and PC3. For complete conversion to insulin, cleavage must occur at both the B-chain/C-peptide and C-peptide/A-chain junctions of proinsulin. Studies in vitro have shown the specificity of PC3 for the B-chain/C-peptide junction and that of PC2 for the C-peptide/A-chain junction. In contrast, studies in vivo have suggested that the proinsulin cleavage substrate specificity of these two endoproteases might be more complex. We have now used recombinant adenovirus to overexpress PC2 or PC3 in the rat insulinoma cell line INS. These cells convert proinsulin more slowly than primary pancreatic beta-cells, possibly reflecting their lower levels of PC3. Infection of INS cells with the corresponding recombinant adenovirus led to 5-10-fold and 20-40-fold increases in PC2 and PC3 expression respectively. Recombinant adenovirus is thus an effective tool for expressing proteins at high levels in slow-growing INS cells. Overexpression of either PC2 or PC3 in INS cells led to a striking acceleration of conversion of proinsulin to insulin and to a decreased accumulation of the conversion intermediate des-64.65-split proinsulin (cleaved only at the A-chain/C-peptide junction). There was no detectable accumulation of des-31.32-split proinsulin (cleaved only at the B-chain /C-peptide junction) after overexpression of either enzyme. Taken together, the data indicate that when expressed at high levels, both PC2 and PC3 seem to be able to cleave proinsulin at both its junctions in vivo.