The mutant proinsulin gene was constructed with the codons for A6 and A11 Cys changed to Ser to delete intra-A chain disulfide bond. After expression and purification, the mutations in the protein were further confirmed by amino acid composition. Electrophoretic mobility of the mutant proinsulin is similar to that of human proinsulin, so are the products of tryptic digestions. The mutant proinsulin, which retains its full radioimmuno activity, shows only 5.4% of receptor binding activity of human proinsulin. This suggests that though the intra-A chain disulfide bond disappears, the other two inter-chain disulfide bonds are still correctly paired, and hence the three dimensional structure has not been altered significantly. This intra-chain disulfide bond is essential for insulin displaying its activity.