The phospholipase A2 (PLA2) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA2s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA2s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of PLA2, we have created semisynthetic human group IB PLA2 in which the N-terminal decapeptide is joined with the 13C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA2 joined with a 13C-labeled fragment of group IB PLA2. Infrared spectral resolution of the unlabeled and 13C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA2 has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA2 is more rigid than that of the IB PLA2, but the N-terminal helix is more flexible. Combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA2, which demonstrates remarkable similarity but also distinct differences compared to that of IB PLA2. Correlation is delineated between structural and membrane binding properties of PLA2s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA2s acts as a regulatory domain that mediates interfacial activation of these enzymes.