Fever, a hallmark of disease, is elicited by exogenous pyrogens, that is, cellular components, such as lipopolysaccharide (LPS), of infectious organisms, as well as by non-infectious inflammatory insults. Both stimulate the production of cytokines, such as interleukin (IL)-1β, that act on the brain as endogenous pyrogens. Fever can be suppressed by aspirin-like anti-inflammatory drugs. As these drugs share the ability to inhibit prostaglandin biosynthesis, it is thought that a prostaglandin is important in fever generation. Prostaglandin E₂ (PGE₂) may be a neural mediator of fever, but this has been much debated,^,,,. PGE₂ acts by interacting with four subtypes of PGE receptor, the EP₁, EP₂, EP₃ and EP₄ receptors. Here we generate mice lacking each of these receptors by homologous recombination. Only mice lacking the EP₃ receptor fail to show a febrile response to PGE₂ and to either IL-1β or LPS. Our results establish that PGE₂ mediates fever generation in response to both exogenous and endogenous pyrogens by acting at the EP₃ receptor.