Abstract Formyltetrahydrofolate synthetase purified from Clostridium cylindrosporum and Clostridium acidi-urici exists as a catalytically active tetramer in the presence of specific monovalent cations or as an inactive monomer in their absence. Addition of monovalent cations to the dissociated enzyme causes the monomers to reassociate to the tetramer. The order of cation effectiveness is NH4+ g T1+ g Rb+ ∼ K+ g Cs+ g Na+ ∼ Li+. The rate and the extent of reactivation is influenced by the counter ion. Sulfate and phosphate, for example, stimulate reassociation while thiocyanate, trichloroacetate, and perchlorate completely inhibit the reaction. This effect is attributed to the ability of the anion to alter the iceberg structure of water which exists about exposed hydrophobic residues on the monomer. Increasing the ionic strength of the reaction medium results in an increase in both the rate and the extent of reassociation. Difference spectroscopy experiments suggest that aromatic amino acids are buried during reassociation. An investigation of the kinetics of cation-dependent subunit reassociation indicates that reactivation of monomers from C. cylindrosporum is second order with respect to monomer concentration, while reactivation of C. acidi-urici monomers is a first order process. It is proposed that the rate-determining step in the C. cylindrosporum case is the combination of two subunits to form a dimer; in the C. acidiurici case, a conformational change in the subunit, which is rate-limiting, must occur before reassociation is possible. The presence of MgATP at concentrations near its Michaelis constant greatly increases the rate of reassociation of monomers from C. acidi-urici. Preincubation of the monomers with MgATP results in a change in the kinetic order of cation-dependent reassociation from first to second order. This substrate, by binding to the monomer, evidently produces the conformational alteration required for reactivation. Low concentrations of MgATP have no effect on the reactivation of C. cylindrosporum monomers.