The conformational properties of vasoactive intestinal peptide (VIP) include the N-terminal randomized structure and the C-terminal long alpha-helical structure. We have previously observed that the N-terminal random coil structure plays a crucial role in the receptor-selectivity. Here, to clarify how the formation of the alpha-helix plays a role in its biological functions, we chemically synthesized VIP analogues modified at the C-terminus, mid-chain, and N-terminus of the alpha-helical region, and evaluated the relationship between their alpha-helical contents and their biological activities including relaxant effects on murine stomach and receptor-binding activities. VIP and VIP-(1-27) showed equipotent biological activities with 48% and 50% alpha-helical content, respectively, each of which corresponds to 14 amino acid residues. VIP-(1-26) was 10% and threefold less potent in relaxant and binding activities, respectively, compared with VIP, and its 49% alpha-helical content resulted in 13 residues involved in the alpha-helix. Further truncation from 25 to 21 resulted in decrease in the alpha-helical content from 43% to 29%, corresponding residues from 11 to 6, the relaxant activity from 72% to 4%, and the affinity to the membrane from 60-fold to over 10(4)-fold less potency. In addition, disruption of the mid-chain and the N-terminus in the alpha-helical stretch by oxidation of Met(17) and deletion of Thr(11) also inhibited biological activities. These findings suggest that the presence of alpha-helical structure forming in 14 amino acid residues between position 10 and 23 in VIP is essential to its biological functions and the C-terminal amino acid residues between position 24 and 27 are requisite for this alpha-helical formation.