The thiobarbituric acid reacting material produced during enzymatic microsomal lipid peroxidation has been identified as malonaldehyde. Malonaldehyde was condensed with urea to form 2-hydroxy-pyrimidine, which was identified by its ultraviolet spectrum, chromatographic properties, and mass spectrum. Incubations with phosphatidyl choline labelled with tritiated arachidonate yielded 2-hydroxy-pyrimidine with a specific activity nearly equal to that of the phospholipid arachidonate. Incubations with tritiated arachidonic acid yielded 2-hydroxy-pyrimidine with a specific activity nearly 2 orders of magnitude less than that of free arachidonic acid. Therefore, phospholipid arachidonate has been established as the major source of the malonaldehyde produced during microsomal lipid peroxidation. During the peroxidation of unsaturated fatty acids or tissue lipids, a substance is produced which reacts with thiobarbituric acid to yield a pink colour. This substance has been generally assumed to be malonaldehyde. Photolysis of linolenic acid has also been shown to give thiobarbituric acid reacting products, which have been reported not to include malonaldehyde. Kwon and Olcott suggested that these other products are artifacts produced during the isolation procedure, and presented chromatographic evidence for the identity of the thiobarbituric acid reacting material with malonaldehyde. Hochstein and Ernst have demonstrated a NADPH dependent enzymatic lipid peroxidation in liver microsomes, which produced a thiobarbituric acid-reacting product presumed to be malonaldehyde. May et al. showed that after NADPH dependent lipid peroxidation there was a decrease in the content of arachidonic and docosahexaenoic acids in the microsomal phospholipids, and a more polar phospholipid fraction appeared. They concluded that these polyunsaturated fatty acids are the source of the thiobarbituric acid reacting material. Using a method recently developed, we have identified the thiobarbituric acid reacting material produced during NADPH dependent lipid peroxidation as malonaldehyde, and have shown that the arachidonic acid of the tissue phospholipid is the major source of the malonaldehyde.The thiobarbituric acid reacting material produced during enzymatic microsomal lipid peroxidation has been identified as malonaldehyde. Malonaldehyde was condensed with urea to form 2-hydroxy-pyrimidine, which was identified by its ultraviolet spectrum, chromatographic properties, and mass spectrum. Incubations with phosphatidyl choline labelled with tritiated arachidonate yielded 2-hydroxy-pyrimidine with a specific activity nearly equal to that of the phospholipid arachidonate. Incubations with tritiated arachidonic acid yielded 2-hydroxy-pyrimidine with a specific activity nearly 2 orders of magnitude less than that of free arachidonic acid. Therefore, phospholipid arachidonate has been established as the major source of the malonaldehyde produced during microsomal lipid peroxidation. During the peroxidation of unsaturated fatty acids or tissue lipids, a substance is produced which reacts with thiobarbituric acid to yield a pink colour. This substance has been generally assumed to be malonaldehyde. Photolysis of linolenic acid has also been shown to give thiobarbituric acid reacting products, which have been reported not to include malonaldehyde. Kwon and Olcott suggested that these other products are artifacts produced during the isolation procedure, and presented chromatographic evidence for the identity of the thiobarbituric acid reacting material with malonaldehyde. Hochstein and Ernst have demonstrated a NADPH dependent enzymatic lipid peroxidation in liver microsomes, which produced a thiobarbituric acid-reacting product presumed to be malonaldehyde. May et al. showed that after NADPH dependent lipid peroxidation there was a decrease in the content of arachidonic and docosahexaenoic acids in the microsomal phospholipids, and a more polar phospholipid fraction appeared. They concluded that these polyunsaturated fatty acids are the source of the thiobarbituric acid reacting material. Using a method recently developed, we have identified the thiobarbituric acid reacting material produced during NADPH dependent lipid peroxidation as malonaldehyde, and have shown that the arachidonic acid of the tissue phospholipid is the major source of the malonaldehyde.