The sterile α-motif (SAM), a relatively small (∼70 amino acids) interaction domain, is found in a variety of proteins involved in cell signaling, transcription regulation, and scaffolding. The Ste11 protein kinase from the mitogen activated protein kinase (MAPK) signaling cascades of the budding yeast is regulated by a SAM domain located at the N-terminus of full-length protein. In solution, the Ste11 SAM domain exists as a well-folded dimeric structure that is involved in interaction with the cognate SAM domain from an adaptor protein Ste50. In this work, we show that the Ste11 SAM domain has an intrinsic affinity towards the lipid membranes. The solution conformation of the Ste11 SAM determined in perdeuterated DPC micelle, using NMR spectroscopy, is defined by five helices of different lengths connected by a number of loops. In the micelle bound state, the non-polar and aromatic residues of the Ste11 SAM lack a native-like packing and are presumably engaged in interactions with the micelle. Using two different paramagnetic doxyl-lipids; we have mapped out localization of Ste11 SAM residues at the micelle surface. Most of the residues appear to localize at the interfacial region of the micelle. However, a number of non-polar residues from the central region of the domain are found to be located inside the core of the micelle including residues from the helix 4 and a loop between helix 2 and helix 3. Isothermal titration calorimetry studies demonstrate that a facile insertion of the Ste11 SAM into the DPC micelle is primarily driven by a large change in enthalpy, −50 kcal/mol with an apparent equilibrium association constant (Ka) of 7.86 × 106 M−1. Interestingly, an interfacial mutant L60R of the Ste11 SAM lacking the dimeric structure does not show detectable interactions with the lipid micelle. The micelle-bound structure of the Ste11 SAM domain described in this work may have potential implications in the regulation of MAPK signaling whereby positioning of the Ste11 protein in close proximity to the membrane may facilitate efficient phosphorylation of the Ste11 kinase by the membrane attached upstream Ste20/pak kinase. Proteins 2009. © 2008 Wiley-Liss, Inc.