Conversion of rat proinsulins I and II was slower in transformed INS cells than in primary (islet) beta cells, with accumulation of des-64,65 but no detectable des-31,32-split proinsulin, indicating slow cleavage at the B-chain/connecting peptide (C-peptide) junction. Western blot analysis showed lower levels of the endoprotease PC1/3 in INS cells than in beta cells, as well as a 4-fold reduction in the ratio of PC1/3 to PC2, thus supporting the hypothesis that PC1/3 is the endoprotease responsible for cleavage at the B-chain/C-peptide junction.