Abstract The ternary complexes of Mn2+-yeast pyruvate kinase formed upon addition of substrates, products, and the activator fructose 1,6-diphosphate were investigated by proton relaxation rate measurements and kinetic studies. As with the muscle enzyme, the enhancement values of the ternary complexes of the yeast enzyme (et) were all less than that of the binary enzyme-Mn2+ complex (eb) suggesting the formation of enzyme-metal-substrate bridge complexes. With the yeast enzyme, ATP binds 13 times more tightly than ADP and et (ADP) l et (ATP). The latter finding indicates conformational changes in addition to water replacement on the enzyme-bound Mn2+. The et (P-enolpyruvate) with the yeast and muscle enzymes are indistinguishable suggesting a similar environment for Mn2+ in the P-enolpyruvate ternary complex. The allosteric activator fructose 1,6-diphosphate has no effect on et (P-enolpyruvate) with yeast pyruvate kinase, but causes a 3-fold decrease in the dissociation constant, in agreement with kinetic data. The dissociation constant (K2) of MnATP is seven times lower than its kinetic inhibition constant (Ki), consistent with either multiple modes of binding of ATP or an ordered dissociation of products with ATP preceding pyruvate. Similarly the dissociation constant for ADP was an order of magnitude lower than its Michaelis constant arguing against a rapid equilibrium random kinetic scheme. Fructose 1,6-diphosphate has little or no effect on the binding of ADP as observed kinetically or by water proton relaxation rate measurements. The homotropic effect of ADP observed kinetically at low Mn2+ concentration is abolished, however, by high concentration of Mn2+ or by the combined interaction of Mn2+ and P-enolpyruvate. Fructose 1,6-diphosphate abolishes the homotropic effect of P-enolpyruvate and decreases the dissociation constant of yeast pyruvate kinase for both P-enolpyruvate and pyruvate but not for FPO32-. It is suggested that the allosteric activator functions primarily by modifying the carboxyl subsite of P-enolpyruvate and thereby raises the affinity of the enzyme for the substrate.