The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D9k (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca2+, the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca2+-loaded calbindin D9k does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca2+ is bound to the protein. A reduction in pH in the absence of Ca2+ increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca 2+ affinity causes a transition to the micelle-associated state also in the presence of Ca2+ when the pH is lowered. Site-specific analysis of the data indicates that calbindin D9k has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca2+-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D9k in the cell is discussed, along with the broader implications for the function of the S100 protein family. textcopyright 2005 American Chemical Society.