Activities of the enzymes involved in cellular respiration are markedly influenced by the composition of the phospholipid environment of the inner mitochondrial membrane. Contrary to previous suppositions, we show that fusion of mitochondria isolated from healthy cardiac muscle with cardiolipin or dioleoylphosphatidylcholine results in a 2-6-fold reduction in the activity of complexes I, II, and IV. The activity of complex III was unaffected by increased phospholipid levels. Phospholipid content had an indiscriminate yet detrimental effect on the combined activities of complexes I+III and II+III. These results have strong implications for therapeutic lipid replacement strategies, in which phospholipid modification of the mitochondria is proposed to enhance mitochondrial function.