Recently, it has been shown that the Toll-like receptors (TLRs)-2 and -6 agonist fibroblast-stimulating lipopeptide-1 (FSL-1) has the capacity to induce fever and sickness behavior in rats. Since the mechanisms of the fever-inducing effects of FSL-1 are still unknown, we tested the pyrogenic properties of FSL-1 in guinea pigs and assessed a role for tumor necrosis factor- (TNF) and prostaglandins in the manifestation of the febrile response to this substance. Intra-arterial (i.a.) and intraperitoneal (i.p.) injections of FSL-1 caused dose-dependent fevers, which coincided with elevated plasma levels of TNF and interleukin-6 (IL-6), the i.p. route of administration being more effective than the i.a. route. I.a. or i.p. injection of a soluble form of the TNF type 1 receptor (referred to as TNF binding protein, TNFbp) together with FSL-1 completely neutralized FSL-1-induced circulating TNF, and reduced fever and circulating IL-6. I.a. or i.p. injection of the non-selective cyclooxygenase (COX)-inhibitor diclofenac depressed fever and FSL-1-induced elevations of circulating prostaglandin E2 (PGE2). Circulating TNF and IL-6, however, remained unimpaired by treatment with diclofenac. In conclusion, FSL-1-induced fever in guinea pigs depends, in shape and duration, on the route of administration and is to a high degree mediated by pyrogenic cytokines and cyclooxygenase products. Mycoplasma infection; diacylated mycoplasmal lipopeptides; cytokines; prostaglandins