Estrogens reduce lipocalin-type prostaglandin D synthase (L-PGDS) expression in a region-dependent manner in the mouse preoptic area (POA). This result linked sex hormones with sleep-wake cycle regulation. In turn, the somnogenic effects of prostaglandin D(2) have been shown to be mediated via increases in adenosine, and a select group of sleep-active ventrolateral preoptic nucleus (VLPO) neurons are directly activated by adenosine 2A (A(2A)) agonists. We hypothesized that increased arousal after estrogen administration is mediated by a reduction of L-PGDS and lowered A(2A) receptor expression in the POA. To test this hypothesis, running wheel activity (RWA) of ovariectomized female mice treated with oil or different doses of estradiol benzoate (EB) was studied, followed by quantitative reverse-transcriptase polymerase chain reaction to determine mRNA expression of genes related to sleep and arousal in brain region extracts from oil-treated and EB-treated mice. RWA was increased in estrogen-treated mice, and these effects followed an inverted-U dose-response curve. The most effective dose (1.25 microg EB/capsule) increased RWA more than 2.5-fold, as compared with control animals, and EB doses that were higher or lower were less effective. Increases in RWA were accompanied by decreased L-PGDS mRNA in the POA and decreased A(2A) receptor mRNA in the POA and VLPO. Given that EB-treated animals have higher motor activity and lower levels of L-PGDS and A(2A) receptor mRNAs in sleep-active areas, these correlational findings support the hypothesis that EB may increase behavioral arousal by decreasing the levels of well-known sleep-inducing molecules within the preoptic region.