The objective of this study was to identify the products and possible role of a putative pathway for de novo fatty acid synthesis in mammalian mitochondria. Bovine heart mitochondrial matrix preparations were prepared free from contamination by proteins from other subcellular components and, using a combination of radioisotopic labeling and mass spectrometry, were shown to contain all of the enzymes required for the extension of a 2-carbon precursor by malonyl moieties to saturated acyl-ACP thioesters containing up to 14 carbon atoms. A major product was octanoyl-ACP and, in the presence of the apo-H-protein of the glycine cleavage complex, the newly synthesized octanoyl moieties were translocated to the lipoylation site on the acceptor protein. These studies demonstrate that one of the functions of the de novo fatty acid biosynthetic pathway in mammalian mitochondria is to provide the octanoyl precursor required for the essential protein lipoylation pathway.