We have recently developed a method to produce native human proinsulin using a bacterial expression system. A proinsulin fusion protein was recovered from inclusion bodies and cleaved using cyanogen bromide. The released proinsulin polypeptide was S-sulfonated and purified by anion exchange chromatography. Following refolding, proinsulin was purified by reversed-phase high-performance liquid chromatography. Combined peptide mapping and mass spectrometric analysis indicated that the proinsulin contained the correct disulfide bridging pattern. This proinsulin will be used to study the specificity of the furin/PC family of converting enzymes by using it as a substrate in a recently developed assay.