We examined the signaling route for fever during localized inflammation in male and female mice, elicited by casein injection into a preformed air pouch. The localized inflammation gave rise to high concentrations of prostaglandins of the E species (PGE(2)) and cytokines in the air pouch and elevated levels of these inflammatory mediators in plasma. There were also elevated levels of PGE(2) in the cerebrospinal fluid, although there was little evidence for PGE(2) synthesis in the brain. Global deletion of the PGE(2) prostaglandin E receptor 3 (EP3) abolished the febrile response as did deletion of the EP3 receptor in neural cells, whereas its deletion on peripheral nerves had no effect, implying that PGE(2) action on this receptor in the CNS elicited the fever. Global deletion of the interleukin-1 receptor type 1 (IL-1R1) also abolished the febrile response, whereas its deletion on neural cells or peripheral nerves had no effect. However, deletion of the IL-1R1 on brain endothelial cells, as well as deletion of the interleukin-6 receptor a on these cells, attenuated the febrile response. In contrast, deletion of the PGE(2) synthesizing enzymes cyclooxygenase-2 and microsomal prostaglandin synthase-1 in brain endothelial cells, known to attenuate fever evoked by systemic inflammation, had no effect. We conclude that fever during localized inflammation is not mediated by neural signaling from the inflamed site, as previously suggested, but is dependent on humoral signaling that involves interleukin actions on brain endothelial cells, probably facilitating PGE(2) entry into the brain from the circulation and hence representing a mechanism distinct from that at work during systemic inflammation.