All phases of lipopolysaccharide (LPS)-induced fever are mediated by prostaglandin (PG) E2. It is known that the second febrile phase (which starts at approximately 1.5 h post-LPS) and subsequent phases are mediated by PGE2 that originated in endotheliocytes and perivascular cells of the brain. However, the location and phenotypes of the cells that produce PGE2 triggering the first febrile phase (which starts at approximately 0.5 h) remain unknown. By studying PGE2 synthesis at the enzymatic level, we found that it was activated in the lung and liver, but not in the brain, at the onset of the first phase of LPS fever in rats. This activation involved phosphorylation of cytosolic phospholipase A2 (cPLA2) and transcriptional up-regulation of cyclooxygenase (COX)-2. The number of cells displaying COX-2 immunoreactivity surged in the lung and liver (but not in the brain) at the onset of fever, and the majority of these cells were identified as macrophages. When PGE2 synthesis in the periphery was activated, the concentration of PGE2 increased both in the venous blood (which collects PGE2 from tissues) and arterial blood (which delivers PGE2 to the brain). Most importantly, neutralization of circulating PGE2 with an anti-PGE2 antibody both delayed and attenuated LPS fever. It is concluded that fever is initiated by circulating PGE2 synthesized by macrophages of the LPS-processing organs (lung and liver) via phosphorylation of cPLA2 and transcriptional up-regulation of COX-2. Whether PGE2 produced at the level of the blood-brain barrier also contributes to the development of the first phase remains to be clarified.