HspA1A, a 70-kDa heat shock protein, binds to specific lipids. This interaction allows HspA1A to associate with the plasma and other cellular membranes, where it regulates many vital functions like immunity, membrane stabilization, autophagy, and apoptosis. However, the molecular mechanism of the HspA1A-lipid interactions has yet to be fully characterized. Therefore, in this study, we characterized the interaction of HspA1A with three lipids, bis-(monoacylglycero)-phosphate, cardiolipin, and sulfatide. Our results revealed that, first, HspA1A embeds in membranes when bound to liposomes composed of cardiolipin and sulfatide. Second, the binding of HspA1A to lipids is complex and although important, electrostatic interactions alone cannot fully explain the observed binding. Third, the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, differentially bind to lipids in a lipid-specific manner. Fourth, HspA1A lipid-binding is reduced by the presence of nucleotides, but it is unaffected by the presence of a peptide-substrate. These observations suggest that HspA1A binds to lipids via a multi-step mechanism and this interaction depends on the specific physicochemical properties of the lipid. We speculate that the association of HspA1A with lipids like the mitochondrial cardiolipin, which is an organelle marker, may facilitate the translocation and localized function of the molecular chaperone to particular sub-cellular compartments. (C) 2015 Elsevier Inc. All rights reserved.