The occupancy of the central cavity of the membrane-embedded c ring of the ATP synthase of Escherichia coli was investigated with a photo-cross-linking approach. Single cysteine mutants were created at c subunit positions 4, 8, and 11, which are oriented to the inside of the ring. These cysteines were alkylated with reagents carrying a photoactivatable substituent and illuminated. Subunit c and derivatives were then isolated and subjected to mass spectrometric analyses. The most noticeable product, which was found exclusively in irradiated samples, had a mass increase of 719 Da, consistent with a cross-link product between the substituted c subunit and phosphatidylethanolamine. Digestion with phospholipase C converted this product into one with a mass diminished by 126 Da, indicating that the phosphoethanolamine moiety was cleaved off. Hence, the cross-link forms to the diacylglycerol moiety of phosphatidylethanolamine. Control experiments showed that the subunit c-phospholipid adducts were formed in the ATP synthase complex in its natural membrane environment and were not artifacts arising from monomeric c subunits. We conclude therefore that the inner lumen of the c ring is occupied with phospholipids. No evidence was found for an extension of subunit a into this space.