To understand how female sex hormones influence homeostatic mechanisms of sleep, we studied the effects of estradiol (E2) replacement on c-Fos immunoreactivity in sleep/wake-regulatory brain areas after sleep deprivation (SD) in ovariectomized rats. Adult rats were ovariectomized and implanted subcutaneously with capsules containing 17-E₂ (10.5 µg; to mimic diestrus E₂ levels) or oil. After two weeks, animals with E₂ capsules received a single subcutaneous injection of 17-E₂ (10 µg/kg; to achieve proestrus E₂ levels) or oil; control animals with oil capsules received an oil injection. Twenty-four hours later, animals were either left undisturbed or sleep deprived by "gentle handling" for 6 h during the early light phase, and killed. E₂ treatment increased serum E₂ levels and uterus weights dose-dependently, while attenuating body weight gain. Regardless of hormonal conditions, SD increased c-Fos immunoreactivity in all four arousal-promoting areas and four limbic and neuroendocrine nuclei studied, whereas it decreased c-Fos labeling in the sleep-promoting ventrolateral preoptic nucleus (VLPO). Low and high E₂ treatments enhanced the SD-induced c-Fos immunoreactivity in the laterodorsal subnucleus of the bed nucleus of stria terminalis and the tuberomammillary nucleus, and in orexin-containing hypothalamic neurons, with no effect on the basal forebrain and locus coeruleus. The high E₂ treatment decreased c-Fos labeling in the VLPO under non-deprived conditions. These results indicate that E₂ replacement modulates SD-induced or spontaneous c-Fos expression in sleep/wake-regulatory and limbic forebrain nuclei. These modulatory effects of E₂ replacement on neuronal activity may be, in part, responsible for E₂'s influence on sleep/wake behavior.