AIM:To study the relationship between insulin A chain regions and insulin biological activities, we designed a series of insulin analogues with changes at A21, A12-18 of C terminal helical region and A8-10 located in the region of A6-A11 intra-chain disulphide bond.METHODS:Insulin A-chain analogues were prepared by stepwise Fmoc solid phase manual synthesis and then combined with natural B-chain of porcine insulin to yield corresponding insulin analogues. Their biological activities were tested by receptor binding, mouse convulsion and immunological assay.RESULTS: A21Ala Ins retains 70.3% receptor binding capacity and 60% in vivo biological activity.DesA13-14, A21Ala Ins and DesA12-13-14-15, A21Ala Ins still have definite biological activity,7.9% and 4.0% receptor binding,and 6.2% and 3.3% in vivo biological activity respectively. A15Asn, A17Pro, A21Ala Ins maintains 10.4% receptor binding and 10% in vivo biological activity. A8His, A9Arg, A10Pro, A21Ala Ins, A8His, A9Lys, A10Pro, A21Ala Ins and A8His, A9Lys, A10Arg, A21Ala Ins have 51.9%, 44.3% and 32.1% receptor binding respectively,50%, 40% and 30% in vivo biological activity respectively, and 28.8%, 29.6% and 15.4% immunological activity respectively.CONCLUSION:A21Asn can be replaced by simple amino acid residues.The A chains with gradually damaged structural integrity in A12-18 helical region and the demolition of the A12-18 helical region by the substitution of Pro and Asn for A17Glu and A15Gln respectively can combine with the B chain and the combination products show definite biological activity, the helical structure of A12-18 is essential for biological activities of insulin. A8-10 is not much concerned with biological activities, but is much more important antigenically in binding to its antibodies, these results may help us design a new type of insulin analogue molecule.