Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent lethal periods of ischemia (ischemic tolerance). In this study, we used NADPH-diaphorase histochemistry to investigate the postischemic changes of nitric oxide synthase (NOS) in the hippocampus in a rat model of cerebral ischemia and ischemic tolerance. Forebrain ischemia was induced by 4-vessel occlusion for 3 min as an ischemic preconditioning. Three days after the preconditioning or sham operation, second ischemia was induced for 6 min. A transient increase in NADPH-diaphorase activity, beginning after 2 h and maximal after 1 day, was observed in CA1 pyramidal neurons of rats subjected to 3 min of preconditioning ischemia as well as 6 min of subsequent ischemia both with and without preconditioning. In addition, expression of NADPH-diaphorase activity was seen in reactive glial cells in the damaged CA1 region of animals subjected to 6 min of ischemia without preconditioning. Thus, direct involvement of increased NADPH-diaphorase activity in ischemic tolerance was not suggested because the increased NADPH-diaphorase activity preceded the induction of ischemic tolerance which takes place 1-7 days after preconditioning. However, the present findings suggest that the induction of neuronal NADPH-diaphorase activity occurs in response to cerebral ischemia.