Cytochrome oxidase isolated from beef-heart mitochondria has been reconstituted into DMPG bilayers at various enzyme/DMPG ratios. In addition, fractions of these samples were incubated with excess cytochrome c in buffer. Membrane complexes were then centrifuged to study lipid-protein interactions at 36 °C by spin label electron spin resonance (ESR) spectroscopy using phosphatidylglycerol spin-labeled at position 14 of the sn-2 acylchain (14-PGSL). For comparison, ESR spectra of this spin-label were recorded for model membranes without cytochrome oxidase but with cytochrome c. All samples were finally analyzed for lipid, cytochrome oxidase and cytochrome c content. It was found that the motionally restricted spin-label component in reconstituted cytchrome oxidase/DMPG bilayers corresponds to 55-60 immobilized lipids per cytochrome oxidase molecule inde pendent of the total amount of DMPG per cytochrome oxidase used in a particular sample. Cytochrome c alone did not induce a second component in 14-PGSL spectra in pure DMPG bilayers at 36 ˚C. However, when cytochrome c was added to reconstituted cytochrome oxidase/DMPG membranes the fraction of motionally restricted spin label increased on binding of cytochrome c indicating structural changes in the membrane. Depending on the DMPG / cytochrome oxidase ratio, up to 115 immobilized lipid molecules / cytochrome oxidase were found when cytochrome c was present. Furthermore, analysis of the DMPG, cytochrome oxidase and cytochrome c content of all samples revealed that under the chosen conditions cytochrome c binds to ~ 9 negatively charged DMPG molecules independent of the cytochrome oxidase content in reconstituted systems.