In Euglena gracilis a malonyl-CoA-independent fatty acid-synthetic system, in which fatty acids are synthesized directly from acetyl-CoA as both primer and C2 donor, occurs in mitochondria, and the system contributes to the wax ester fermentation. The activity of fatty acid synthesis in the mitochondrial system was enhanced about six times when an artificial acetyl-CoA-regenerating system was present, indicating that the fatty acid-synthetic activity is controlled by the ratio of acetyl-CoA against CoA. When fatty acids were synthesized using pyruvate instead of acetyl-CoA as substrate, a high activity, about 30 times higher than that from acetyl-CoA, was found under anaerobic conditions (below 10(-5) M oxygen), while in aerobiosis fatty acids were not synthesized at all. CoA, NADH, and NADP+ were required as cofactors for fatty acid synthesis from pyruvate. It was indicated that high activity of fatty acid synthesis from pyruvate due to the high ratio of acetyl-CoA against CoA was maintained by the action of the oxygen-sensitive pyruvate dehydrogenase found in Euglena mitochondria. When [2-14C]pyruvate was fed into intact mitochondria under anaerobic conditions, radioactive fatty acids were formed in the presence of malate, which provided reducing power for the matrix.