Tension-induced exposure of a cryptic signaling binding site is one of the most fundamental mechanisms in molecular mechanotransduction. Helix bundles in rod domains of talin, a tension-sensing protein at focal adhesions, unfurl under tension to expose cryptic vinculin binding sites. Although the difference in their mechanical stabilities would determine which helix bundle is tension-sensitive, their respective mechanical behaviors under tension have not been characterized. In this study, we evaluated the mechanical behaviors of residues 486-654 and 754-889 of talin, which form helix bundles with low and high tension-sensitivity, by employing AFM nano-tensile testing. As a result, residues 754-889 exhibited lower unfolding energy for complete unfolding than residues 486-654. In addition, we found that residues 754-889 transition into intermediate conformations under lower tension than residues 486-654. Furthermore, residues 754-889 showed shorter persistence length in the intermediate conformation than residues 486-654, suggesting that residues 754-889 under tension exhibit separated alpha-helices, while residues 486-654 assume a compact conformation with inter-helix interactions. Therefore, we suggest that residues 754-889 of talin work as a tension-sensitive domain to recruit vinculin at the early stage of focal adhesion development, while residues 486-654 contribute to rather robust tension-sensitivity by recruiting vinculin under high tension. (C) 2017 Elsevier Inc. All rights reserved.