The current study was performed to determine the role of reactive oxygen species (ROS) in preconditioning against different forms of neuronal damage. Primary cultures of chick embryonic neurons were treated with either FeSO(4) (100 microM; 15 min) to generate hydroxyl radicals or xanthine/xanthinoxidase (10 microM/0.5 mU ml(-1); 15 min; =X/XO (pre)) to produce superoxide radicals. Both stimuli moderately enhanced ROS formation as measured by fluorescence microscopy. This preconditioning significantly protected the neurons against subsequent glutamate (1 mM)-induced excitotoxic damage, staurosporine (200 nM)-induced neuronal apoptosis and oxidative damage caused by exposure to xanthine/xanthinoxidase (500 microM/5 mU ml(-1); 1 h; =X/XO (dam)). The antioxidants vitamin E (10 microM) and 2-OH-estradiol (1 microM), present during the 15-min preconditioning period, completely abolished the protective effect of X/XO (pre). Furthermore, glutamate, staurosporine or X/XO (dam) markedly enhanced oxygen radical formation. Preceding preconditioning by mild ROS stimulation with X/XO (pre) or Fe(2+) reduced this oxygen radical burst. Again, the effect of X/XO (pre) could be blocked by coadministration of vitamin E or 2-OH-estradiol. However, the FeSO(4)-mediated preconditioning was not abolished by the radical scavengers. To address this phenomenon, the effect of vitamin E and 2-OH-estradiol on Fe(2+)- and X/XO (pre)-induced ROS formation kinetics within the 15 min of preconditioning was monitored. The moderate rise of intracellular ROS content during preconditioning was only reduced permanently by the antioxidants, when the neurons were treated with X/XO (pre), but not when Fe(2+) was used. Thus, an immediate and constant radical scavenging seems to be indispensable to abolish the ROS-induced neuronal preconditioning. The current results indicate that preconditioning by moderate ROS-stimulation protects cultured neurons against different damaging agents and prevents against the subsequent massive oxygen radical formation.