Mitochondrial creatine kinase (MtCK) is a key enzyme for bioenergetics, membrane topology and possibly also for general organelle morphology. X-ray structural analysis (1), EM (2) and mutational studies with SPR (3,4) revealed that the large MtCK octamers bind to and “cross-link” mitochondrial membranes by their two identical top or bottom faces (5). These expose expose four C-terminal basic interaction motifs that interact mainly with acidic cardiolipin (4). This interaction induces cardiolipin-rich domains in the membrane (5,6). However, earlier data point to additional hydrophobic interactions (7,8). Using SPR, we have performed a thermodynamic analysis of the MtCK binding process. Main results were: (i) Affinity of the MtCK-cardiolipin interaction increases with temperature, pointing to a participation of hydrophobic interactions. (ii) Rate constants of two MtCK binding sites identified earlier differed in temperature-dependence. (iii) Thermodynamic parameters revealed that the gain in free energy of MtCK binding mainly depends on the contribution of entropy, possibly due to charge neutralization and release of bound water. These data are consistent with a two-phase model of rapid electrostatic docking of MtCK to cardiolipin, and slower anchoring via a C-terminal hydrophobic MtCK stretch. This would reinforce MtCK membrane interaction, allow integration of this bulky enzyme into the narrow mitochondrial intermembrane space, and contribute to its functional coupling with adenine nucleotide translocator.1. Eder M et al (2000) Proteins 39, 216.2. Schlattner et al (2000) Biol Chem 381, 1063.3. Schlattner et al (2000) J Biol Chem 275, 17314.4. Schlattner et al (2004) J Biol Chem 279, 24334.5. Schlattner et al (2009) Biochim Biophys Acta 1788, 2032.6. Epand et al (2007) J Mol Biol 365, 968.7. Rojo et al (1991) FEBS Lett 281, 123.8. Granjon et al (2001) Biochemistry 40, 6016.