The synthesis of Fos, the protein product of the immediate early gene c- fos, was used to map metabolically some of the neural substrates of conditioned fear in the rat. Analysis of the behaviors emitted by the rats during the test session provided strong evidence that the conditioning procedure was effective. Exposure to the environment in which they had previously received footshock significantly increased the number of Fos-like immunoreactive neurons in nearly 50 brain regions, both cortical and subcortical. Among the structures showing the most dramatic increases in fear-induced c-fos expression were the cingulate, piriform, infralimbic, and retrosplenial cortices, the anterior olfactory nucleus, claustrum, endopiriform nucleus, nucleus accumbens shell, lateral septal nucleus, various amygdalar nuclei, paraventricular thalamic nucleus, ventral lateral geniculate nucleus, the ventromedial, lateral, and dorsal hypothalamic nuclei, the ventral tegmental area, and the supramammillary area. These data demonstrate that a relatively simple classical conditioning procedure activates a large number of widely dispersed cortical and subcortical structures. Some of the structures showing increased c-fos expression have important autonomic functions and may therefore have reflected centrally mediated changes in blood pressure and respiration produced by the anxiogenic stimuli. In a second experiment, the effects of pretreatment with the anxiolytic drug diazepam (2.5, 5.0, or 10 mg/kg) were evaluated. The benzodiazepine produced dose-related decreases in the frequency of crouching (freezing) elicited by the aversively conditioned contextual cues. Diazepam also produced dose-related decreases in conditioned stress-induced c-fos expression in all but one structure, the effects being statistically significant in 38 of 60 sampled structures. Diazepam dose dependently increased fear-induced c- fos expression in the central nucleus of the amygdala. There was considerable regional variability with respect to sensitivity to diazepam, the retrosplenial cortex and the supramammillary area being the only two structures to show decreases after the lowest dose of diazepam. In contrast, the entorhinal cortex, nucleus accumbens core, ventromedial and posterior hypothalamic nuclei, median raphe, and locus coeruleus were particularly resistant to diazepam, all failing to show statistically significant decreases in conditioned fear-induced c-fos expression even at the highest dose. The extent to which diazepam decreased conditioned stress-induced c-fos expression was unrelated to previous estimates of benzodiazepine receptor density in the sampled structures.