The conformational preferences of AnxI(N26), a peptide corresponding to residues 2-26 of human annexin I, were investigated using CD and NMR spectroscopy. CD results showed that AnxI(N26) adopts a mainly alpha-helical conformation in membrane-mimetic environments, TFE/water and SDS micelles, while a predominantly random structure with slight helical propensity in aqueous buffer. The helical region of AnxI(N26) showed a nearly identical conformation between in TFE/water and in SDS micelles, except for the orientation of the Trp-12 side-chain, which was quite different between the two. The N-terminal region of the AnxI(N26) helix showed a typical amphipathic nature, which could be stabilized by the neighboring hydrophobic cluster. The helical stability of the peptide in SDS micelles was increased by addition of calcium ions. These results suggest that the N-terminal tail domain of human annexin I interacts with biological membranes in a partially calcium-dependent manner.