Proinsulin is usually targetted to the regulated secretory pathway of beta cells, and converted to insulin in beta granules. Under certain pathological situations, a significant amount of proinsulin becomes diverted to the constitutive pathway. To study the kinetics of proinsulin conversion in the constitutive pathway, FAO (hepatoma) cells, which secrete proteins uniquely via this pathway and not the regulated pathway, were stably transfected with cDNA encoding human, rat I or rat II proinsulin. Products released to the medium of transfected cells were analysed by reversed phase HPLC and radioimmunoassay. For human proinsulin, des 31,32 split proinsulin (the conversion intermediate resulting from cleavage only at the B-chain/C-peptide junction followed by trimming of C-terminal basic residues by carboxypeptidase) was the only detectable conversion intermediate; for rat proinsulin II it was des 64,65 split proinsulin (cleaved and trimmed only at the C-peptide/A-chain junction); for rat proinsulin I, both intermediates were seen. Complete processing to insulin occurred for all three, but was most extensive for rat proinsulin I. When considered with the corresponding proinsulin sequences, these data show that a -4 basic residue (i.e. 4 residues N-terminal to the site of cleavage) facilitates proinsulin conversion in the constitutive pathway, and that arginine is preferred over lysine.