Mitochondria with good respiratory activities, as judged from polarographic studies of oxygen consumption, measurement of ADP/O and respiratory controls, have been isolated from cauliflower florets and potato tubers. Examination of the centrifugation pellets by means of electron microscopy have shown well‐preserved organelles, with a condensed matrix and a minimal contamination by other membranes. Additional mitochondria pellets have been prepared from germination roots of Vicia faba and Lupinus albus. In these mitochondria, the phosphorylations were not tightly coupled to oxidations. Total fatty acid compositions of the mitochondria isolated from the four different species were not identical: linoleic acid was predominant in potato and Vicia faba root mitochondria; linolenic acid was predominant in cauliflower or Lupinus albus root mitochondria. When isolated mitochondria, from any species, are incubated under aerobic conditions in a suitable solution containing various cofactors (ATP, NADPH, CoASH, Mg2+) and a respiratory substrate (succinate), any one of the following labelled precursors: [1‐14C]acetate, [2‐14C] acetate, [1‐14C]acetyl‐CoA, [2‐14C]malonate or [2‐14C]malonyl‐CoA is incorporated into the mitochondrial fatty acids. The labelled fatty acids are the same, starting from any one of the four precursors, in the same conditions. With tightly coupled mitochondria, the incorporation of the labelled precursor into the mitochondria fatty acids is linear during 60 min, provided the mitochondria are furnished with a respiratory substrate. This is not true with uncoupled mitochondria. The amount of labelled acetate incorporated into the mitochondrial fatty acids increases with increasing quantities of mitochondrial proteins put in the incubation medium. With cauliflower mitochondria, a strict proportionality has been found between the biosynthesis intensities and the quantities of mitochondrial proteins; with the mitochondria of the other species, the incorporation intensities reach a plateau for high protein concentrations. The isolated mitochondria of the four species synthesize the same fatty acids under aerobic conditions, i.e. palmitic, hexadecenoic, stearic and octadecenoic acids. (These newly synthesized fatty acids are analyzed by radio‐gas chromatography.) No significant quantities of polyunsaturated fatty acids are formed. Mono‐unsaturated fatty acids are not synthesized under anaerobic conditions. Dilute mitochondrial suspensions synthesize predominantly palmitic (50%) and octadecenoic acid (40%); concentrated mitochondrial suspensions synthesize predominantly palmitic (50%) and stearic acid (30%). Dilute mitochondrial suspensions synthesize during the first 5 min of incubation exclusively palmitic and palmitoleic (9‐hexadecenoic) acid (identified by radio‐gas chromatography of the products of oxidative cleavage). With longer incubation times, newly synthesized oleic (9‐octadecenoic) acid and cis‐vaccenic (11‐octadecenoic) acid appear in great quantities in the isolated mitochondria, with only trace amounts of stearic acid.