SUMMARY It has been found that extracts of liver and kidney catalyze the hy- drolysis of 2,4-diketovaleric acid, yielding nearly equivalent amounts of pyruvic and acetic acids. This reaction occurs aerobically and anaerobi- cally, and without detectable acetoacetate formation. The effects of substrate and liver extract concentration on the rate of hydrolysis have been studied. Formation of pyruvic acid from 2,4-diketovaleric acid has also been demonstrated in liver slices. A series of eleven other 2,4-diketo acids is hydrolyzed in a similar manner to yield pyruvic acid. The hydroly- sis of 2,4-diketoundecylic acid resulted in the appearance of equimolar amounts of pyruvic and octanoic acids. Of the diketo acids studied, 2,4- diketohexanoic and 2,4-diketocapric acids are hydrolyzed most rapidly. The pH optima for hydrolysis of the normal aliphatic 2,4-diketo acids are in the range 7.2 to 7.9 for the 5-, 6-, and 7-carbon acids, and 8.0 to 8.9 for the acids containing between 8 and 11 carbon atoms. The term “acylpyruvase” is proposed for the enzyme or enzymes which catalyze 2,4-diketo acid hydrolysis. Acylpyruvase activity is present in the liver and kidney of the rat, rabbit, cat, and mouse. All other tissues studied, including primary rat hepatoma, possess little enzymatic activity. A partially purified enzyme preparation has been obtained from rat liver. Methods of preparation of the disodium salts of 2,4-diketo acids are given. The ultraviolet absorption spectra for the ethyl esters of aliphatic and aromatic 2,4-diketo acids and the relationship between enolization and ultraviolet absorption are described. A spectrophotometric method for the determination of acylpyruvase activity is presented. Implications of these findings in terms of intermediary metabolism are discussed.