The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTPγS on the kinetics of cell-free activation of NADPH oxidase in membranes of resting human neutrophils were investigated. Arachidonate-mediated activation of the oxidase followed a first-order reaction course (k(obs.) = 0.39 min-1 at 26°C). In the presence of NaF during the activation process, activity was enhanced while the activation rate was slightly reduced (k(obs.) = 0.25 min-1 at 26°C). Neomycin blocked activation (half-maximal effect at 25 μM) without affecting rates of superoxide release by preactivated enzyme in vitro or in vivo. In spite of reduced specific activity neither the first-order rate constant of the activation nor the K(m) of the oxidase were altered by neomycin. Oxidase activated in the presence of GTPγS exhibited increased specific activity and unchanged K(m); the course of the reaction deviated from first-order kinetics. Kinetic evidence is presented for two separate activation reactions: a GTPγS-independent, basal, first-order process and a GTPγS-dependent sigmoid activation process. The results are compatible with the existence in neutrophil membranes of two separate pools of dormant oxidase. An alternative scheme of the formation of two active forms of NADPH oxidase is also presented.