Sticholysin I (StI), an actinoporin expressed as a water-soluble protein by the sea anemone Stichodactyla helianthus, binds to natural and model membranes, forming oligomeric pores. It is proposed that the first event of a multistep pore formation mechanism consists of the monomeric protein attachment to the lipid bilayer. To date there is no high-resolution structure of the actinoporin pore or other membrane-bound form available. Here we evaluated StI:micelle complexes of variable lipid composition to look for a suitable model for NMR studies. Micelles of pure or mixed lysophospholipids and of dihexanoyl phosphatidylcholine (DHPC) were examined. The StI:DHPC micelle was found to be the best system, yielding a stable sample and good quality spectra. A comprehensive chemical shift perturbation analysis was performed to map the StI membrane recognition site in the presence of DHPC micelles. The region mapped (residues F51, R52, S53 in loop 3; F107, D108, Y109, W111, Y112, W115 in loop 7; Q129, Y132, D134, M135, Y136, Y137, G138 in helix-α2) is in agreement with previously reported data, but additional residues were found to interact, especially residues V81, A82, T83, G84 in loop 5, and A85, A87 in strand-β5. Backbone dynamics measurements of StI free in solution and bound to micelles highlighted the relevance of protein flexibility for membrane binding and suggested that a conformer selection process may take place during protein–membrane interaction. We conclude that the StI:DHPC micelles system is a suitable model for further characterization of an actinoporin membrane-bound form by solution NMR. Proteins 2014; 82:1022–1034. © 2013 Wiley Periodicals, Inc.