The relative potencies of the (+) and (-) isomers of naloxone in antagonizing electrophysiological responses to D-alanine2-methionine enkephalinamide were compared in rat frontal cortex and hippocampus. In the in vitro hippocampus, the (-) isomer was found to be at least a 100 times more potent than the (+) isomer in antagonizing opiate-induced changes in field potentials. Similar stereoselectivity was observed in vivo in both frontal cortex and hippocampus in terms of the antagonism of enkephalin-induced changes in spontaneous cell firing. The direct effects of (+) and (-)-naloxone were examined as well. In hippocampus both in vivo and in vitro, no differential effect was observed, whereas in the neocortex (-)-naloxone was considerably more potent than the (+) isomer in eliciting depressions of spontaneous activity. These direct effects of naloxone in the cortex do not appear to be due to an antagonism of the effects of endogenously released opioids. These results demonstrate that the stereoselectivity of naloxone isomers in antagonizing electrophysiological responses to opiates in the cortex and hippocampus parallels that previously observed in other brain regions and in other tissues. In addition, they suggest that naloxone may have interactions with other unknown opiate (or possibly non-opiate) receptors which are of physiological significance.