Generation of reactive oxygen species (ROS) in synaptosomes was investigated in the presence of different substrates. When pyruvate was used as a substrate an increased rate of hydrogen peroxide formation was detected by the Amplex Red fluorescent assay, but aconitase, which is known to be a highly sensitive enzyme to ROS was not inhibited. In contrast, pyruvate exerted a partial protection on aconitase against a time-dependent inactivation that occurred when synaptosomes were incubated in the absence of substrates. Disruption of synaptosomal membranes with Triton X-100 prevented the protective effect of pyruvate. It is suggested that citrate and/or isocitrate formed in the metabolism of pyruvate could be responsible for a partial protection of aconitase. Therefore while pyruvate could have a prooxidant effect it could also exert a protective effect on the aconitase.