Abstract Procedures are described for the purification of malate synthetase from baker's yeast and from glycollate-grown Pseudomonas ovalis Chester. The properties of the enzymes are closely similar: both re optimally active at pH 8.5 but that from yeast shows greater activity below this pH than that from Ps. ovalis . The K m for glyoxylate was 9.3· 10−5 M (yeast) and 6.3· −5 M ( Pseudomonas ). The K m for acetyl-S-CoA was −5 M . The enymes require Mg ++ for activity ( K m =5·10 −4 M ) and are competitively inhibited by the C 2 -acids oxalate ( K i =1.9·10 −5 M ) fluoroacetate ( K i =2.46·10−4 M ) and glycollate ( K i =3.08·10 −4 M ). The purified enzymes are specific for glyxoxylate, and do not cytalyse the cleavage of acetyl-S-CoA in the presence of oxaloacetate, pyruvate, α-ketoglutarate, glyoxal, glycolaldehyde, formaldehyde, or acetaldehyde. In the presence of glyoxylate, there was no reaction with S,N-diacetylβ-mercaptoethylamine, S-acetyl pantetheine, propionyl coenzyme A or butyryl coenzyme A, but fluoroacetyl A was split at approximately one quarter of the rate observed with similar concentrations of acetyl-S-CoA. Malyl-S-CoA was not split by the enzymes, either alone or in the presence of glyoxylate. The equilibrium of the reaction strongly favours malate formation: no reversal of the reaction was detected. Procedures are described for the preparation of [ 35 S]coenzyme A, which was used to test for the possible formation of an acyl enzyme as an intermediate in the malate synthetase reaction. Then non-enzymic isotopic exchange of 35 S from CoA 35 SH with acetyl-S-Coenzyme A observed was not affected by the presence of malate synthetase. The properties of malate synthetase are compared with those of the citrate-forming condensing enzyme.