The kinetic mechanism of Pseudomonas cepacia salicylate hydroxylase has been examined by steady state initial rate measurements, and stopped flow and equilibrium studies. Results indicate that salicylate and NADH bind to the hydroxylase randomly. The enzyme is reduced and NAD+ is released. Oxygen subsequently binds to the reduced enzyme . substrate complex, leading to the production of hydroxylated product, CO2, and water. Based on results of anaerobic rapid mixing experiments, the rate of enzyme reduction by NADH is enhanced 290- and 240-fold when the hydroxylase is complexed with salicylate and benzoate (a nonsubstrate effector), respectively. Salicylate enhances, whereas benzoate slightly weakens, the NADH binding to the enzyme. Primary isotope effects were observed with (4R)-[4-2H]- and (4R)-[4-3H]NADH but not with the (4S)-[4-2H]NADH. Using varying concentrations of benzoate, the pattern of tritium isotope effect on Vm/Km, T(V/K), also indicates that benzoate and NADH bind to the enzyme randomly. The intrinsic isotope effects, Dk, of (4R)-[4-2H]NADH on the reduction of enzyme . salicylate and enzyme . benzoate complexes were found to be 5.57 and 5.96, respectively. The former is much repressed but the latter is only slightly so in the expression of their corresponding deuterium isotope effects on Vm, DV. Furthermore, values of DV (1.69 to 5.07) show a rough correlation with the extents of uncoupling of substrate hydroxylation and H2O2 formation activities for a series of benzenoid effectors. These results indicate that relative to the step of enzyme reduction, the subsequent reaction(s) leading to H2O2 formation must be fast whereas that for substrate hydroxylation contains at least one slow step.