Conjointly, the solvent-exposed residues of the central alpha-helix of the B chain form a well-defined ridge, which is flanked and partly overlapped by the two described insulin receptor binding surfaces on either side of the insulin molecule. To evaluate the importance of this interface in insulin receptor binding, we developed a new powerful method that allows us to introduce all the naturally occurring amino acids into a given position and subsequently determine the receptor binding affinities of the resulting insulin analogues. The total amino acid scanning mutagenesis was performed at positions B9, B10, B12, B13, B16, and B17, and the vast majority of the insulin analogue precursors were expressed and secreted in amounts close to that of the wild-type (human insulin) precursor. The analogue binding data revealed that positions B12 and B16 were the two positions most affected by the amino acid substitutions. Interestingly, the receptor binding affinities of the B13 analogues were also markedly affected by the amino acid substitutions, suggesting that GluB13 indeed is a part of insulin's binding surface. The B10 library screen generated analogues covering a wide range of (20-340%) of relative binding affinities, and the results indicated that a structural stabilization of the central alpha-helix and thereby a more rigid presentation of the binding epitope at the insulin receptor is important for receptor recognition. In conclusion, systematic amino acid scanning mutagenesis allowed us to confirm the importance of the B chain alpha-helix as a central recognition element serving as a linker of a continual binding surface.