1. 1. We have studied the phosphotransacetylase (acetyl-CoA:orthophosphate acetyltransferase, EC 2.3.1.8) catalysed acylation of coenzyme A (CoASH) by various acyl phosphates in Tris buffers at 25°. ▪ Under the conditions used, the equilibrium position lies well to the right for all the acyl phosphates studied, and our results refer predominently to the forward step. 2. 2. We have established the kinetic form of the acylation. Both substrates, CoASH and acyl phosphate, are adsorbed by the enzyme, probably under pre-equilibrium (Michaelis-Menten) conditions. Kinetic analysis leads to the various substrate dissociation constants, K 8 , and to the rate constant, k 2 , for the transformation of the enzyme-substrate ternary complex to products. 3. 3. The stability of the acyl phosphate-enzyme adduct depends upon the electron availability on the phosphate and carbonyl oxygen atoms, but rather little upon the steric bulk of the group R in the acyl phosphate. The value of k 2 , however, is very dependent on this steric bulk. 4. 4. The product CoASCOR is not taken up by phosphotransacetylase in the presence of an excess of acyl phosphate. The product HPO 4 2− is less strongly absorbed than any of the acyl phosphates studied. 5. 5. Experiments at pH values between 6 and 9 show that the acyl phosphates are taken up by an acidic centre in the enzyme of effective pK a > 9 . The adsorption is assisted by the acid form of a group with pK a = 6.9 in the free enzyme and pK a = 6.4 in the enzyme-acyl phosphate complex. The adsorption of CoASH probably involves its thiol group, which becomes attached to a basic group in the enzyme-acyl phosphate complex of pK a < 6 . Two other groups, one in the enzyme ( pK a = 8.7 ) and the other in the enzyme-CoASH complex ( pK a = 7.2 ), both active in their acid forms, are involved in binding CoASH. 6. 6. The value of k 2 is also pH-dependent. Two groups in the ternary complex, one ( pK a = 7.3 ) active in its basic form, the other ( pK a = 8.7 ) active in its acidic form, are involved. 7. 7. The spontaneous reaction between CoASH and acetyl phosphate in the relevant pH region is very slow. The acceleration produced by phosphotransacetylase constitutes a factor of at least 5 · 10 5 . 8. 8. The spontaneous reaction of aliphatic carboxylic anhyrides with CoASH proceeds at a rate at least 10 8-fold faster than does the spontaneous reaction with acyl phosphates. 9. 9. The mechanism of the enzymatic reaction is discussed and an outline scheme presented which is compatible with all the evidence. An essential role of the enzyme is to bring into close proximity the two reactants, both of which carry more than one negative charge. Multiple, simultaneous, intramolecular acid and base catalysis is also probably involved.