Kinetic studies of L-Alanine dehydrogenase from Bacillus subtilis-catalyzed reactions in the presence of Zn^(2+) were carried out. The substrate (L-alanine) saturation curve is hyperbolic in the absence of the metal ion but it becomes sigmoidal when Zn^(2+) is added to the reaction mixture indicating the positive cooperative binding of the substrate in the presence of zinc ion. The cooperativity of substrate binding depends on the zinc ion concentration: the Hill coefficients (n_H) varied from 1.0 to 1.95 when the zinc ion concentration varied from 0 to 60 μM. The inhibition of AlaDH by Zn^(2+) is reversible and noncompetitive with respect to NAD^+ (K_i = 5.28 × 10^(-5) M). Zn^(2+) itself binds to AlaDH with positive cooperativity and the cooperativity is independent of substrate concentration. The Hill coefficients of substrate biding in the presence of Zn^(2+) are not affected by the enzyme concentration indicating that Zn^(2+) binding does not change the polymerization-depolymerization equilibria of the enzyme. Among other metal ions, Zn^(2+) appears to be a specific reversible inhibitor inducing conformational change through the intersubunit interaction. These results indicate that Zn^(2+) is an allosteric competitive inhibitor and substrate being a non-cooperative per se, excludes the Zn^(2+) from its binding site and thus exhibits positive cooperativity. The allosteric mechanism of AlaDH from Bacillus subtilis is consistent with both MWC and Koshland`s allosteric model.