We examined the utilization of lactate as an energy substrate in ischemic preconditioned slices obtained from the rat brain left hemisphere, of which the contralateral middle cerebral artery was occluded 48 h before the slice preparation. The levels of high-energy phosphates in the brain slices were measured using 31P NMR with a time resolution of 4 min at 25 degrees C. When iodoacetic acid-pretreated brain slices were further treated with fluorocitrate, a glial toxin, for 2 h (neuron-rich slices), the recovery of the phosphocreatine (PCr) level in artificial cerebrospinal fluid (ACSF) containing lactate after high-K+ stimulation was completely abolished in intact slices, whereas the PCr level in ischemic preconditioned slices well recovered in otherwise similar conditions. These results indicated that neurons, when preconditioned with ischemia, acquire the ability to utilize lactate as an energy substrate. In parallel experiments, we recorded population excitatory postsynaptic potentials and spikes from granule cells in hippocampal slices. Population spikes of intact slices in ACSF containing lactate were completely abolished in 30 min, but those of the ischemic preconditioned slices were maintained well over 50%. These results show that ischemic preconditioning may induce certain systematic changes in neurons, such as the expression of lactate transporters and/or the activation of lactate dehydrogenase.