Lipids of cytochrome c oxidase (COX) of Paracoccus denitrificans have been identified by MALDI-TOF/MS direct analyses of isolated protein complexes, avoiding steps of lipid extraction or chromatographic separation. Two different COX preparations have been considered in the present study: the enzyme core consisting of subunits I and II (COX-2SU) and the complete complex comprising all 4 subunits (COX-4SU). In addition, MALDI-TOF/MS lipid profiles of bacterial COX are also compared with those of the isolated mitochondrial COX and bacterial bc1 complex. We show that main lipids associated with bacterial COX-4SU are phosphatidylglycerol (PG) and phosphatidylcholine (PC), plus minor amounts of cardiolipin (CL). PG and PC are absent in the COX-2SU preparation lacking subunits III and IV, whereas cardiolipin is still present. Quantitative analyses indicate that at variance from mitochondrial COX, cardiolipin is present in substoichiometric amounts in bacterial COX, at a CL/COX molar ratio ~1/10. It is concluded that bacterial COX does not require CL, neither for structure nor for its activity.