14C-halothane direct photoaffinity labeling was used to characterize the distribution of halothane binding in rat brain to test the hypothesis that anesthetics bind preferentially to a specific, heterogeneously distributed, receptor or channel. Slide-mounted sagittal rat brain sections were placed in gas-tight quartz cuvettes with 100 microM 14C-halothane in phosphate buffered saline with 0 to 7.5 mM unlabeled halothane, or unlabeled chloroform and isoflurane at 10 times the clinical EC50, and then exposed to UV light for 60 to 100 sec. Autoradiograms of nine brain regions (cortex, corpus callosum, hippocampal molecular and pyramidal layers, dentate molecular and granule cell layers, and cerebellar molecular, granular and white matter layers) were prepared and quantitated using Image 1.44. Total label incorporation was widespread, but exhibited subtle heterogeneity. There was significantly more total labeling in regions of high synaptic density than in regions containing primarily cell bodies or white matter. Most labeling (approximately 80%) was displaced by unlabeled halothane and can therefore be considered specific. Significantly more specific labeling was found in regions of high synaptic density. Isoflurane did not inhibit halothane photolabeling significantly, but chloroform inhibited it by approximately 50%. In conclusion, halothane photolabeling distribution in the mammalian brain is widespread, saturable and selective, but does not mimic the distribution of any individual receptor or channel. Brain regions with high synaptic density displayed the greatest degree of specific binding, consistent with transmission being an important functional target of volatile anesthetics. These results suggest a remarkably widespread individual target, or more likely, similar binding sites in multiple targets, and are consistent with the notion that anesthesia is the result of action at multiple sites.