Peripheral administration of a variety of inflammatory stimuli such as endotoxin or cytokines induces an orchestrated set of brain-mediated events referred to as the sickness response. The mechanism for how immune products signal the brain is not clear, but accumulating evidence supports the existence of neural as well as blood-borne pathways. Although endotoxin or cytokine administration results in sickness responses, little data exist regarding the role of circulating endotoxin or cytokines in the induction of sickness during a real bacterial infection. Thus, the present studies examined if subcutaneously administered E. coli can activate sickness responses and whether circulating endotoxin and/or pro-inflammatory cytokines are a prerequisite for these responses. Male Sprague-Dawley rats were injected subcutaneously with one of three doses (2.5 x 10⁷, 2.5 x 10⁸, 2.5 x 10⁹ CFU) of replicating E. coli, a ubiquitous bacterial strain, or vehicle. Core body temperature (Tc) and activity were measured for 3 days following the injection. A second set of groups of animals were sacrificed 3, 6, 12, 18, 24, and 48 hours after the injection and blood samples and brains were collected. Injections dose-dependently and consistently increased Tc and decreased activity, with increases in Tc beginning 4 hours after the injection. In addition, E. coli significantly increased serum IL-1, IL-6 and TNF-, and brain IL-1 levels beginning at the 6 hour time point. Corticosterone and endotoxin were first elevated in the circulation at 3 and 18 hours after the injection, respectively. Because fever onset preceded brain cytokine induction, we also examined cytokine levels in the serum, brain, and inflammation site 2 and 4 hours after injection. Cytokines were elevated at the inflammation site, but not detectable in the serum or brain at 2 and 4 hours. We conclude that subcutaneous injection of replicating E. coli induces a consistent and naturalistic infection that includes features of the sickness response as well as increases in circulating, brain and inflammation site tissue cytokines. In addition, injection of replicating E. coli produces a robust fever and corticosterone response at a time when there are no detectable increases in circulating cytokines or endotoxin. These results suggest that elevated levels of circulating cytokines and endotoxin are not necessary for the activation of the sickness or corticosterone response. Therefore, fever, activity reduction, and corticosterone elevation induced by E. coli infection may have been evoked by a neural, rather than a humoral, pathway from the periphery to the brain.