Met-Lys-human proinsulin could be converted into insulin in vitro with the treatment of trypsin and carboxypeptidase B(CPB). Under less effective conditions, the enzymatic reaction does not proceed perfectly, and two main bands have been identified by native-polyacrylamide gel electrophoresis (PAGE) analysis. These two main products were thus separated and purified by DEAE-Sephadex A25 chromatography in a Tris-isopropanol system with an NaCl gradient. The isopropanol and NaCl were removed by a second DEAE-Sephadex column. Native-PAGE, mass spectrometric, and amino acid composition analyses indicate that one fraction of these two major products contains human insulin and desB30-insulin and that the other fraction is a mixture of human insulin analogs, which have one more basic amino acid than human insulin owing to the unsuitable amount of proteases, especially the lack of CPB. Furthermore, both receptor binding assay and radioimmunoassay have been utilized for the activity determination, and both fractions display almost full biological activity with porcine insulin as the standard. Present results provide further evidence for the quality control of recombinant human insulin production.