Toll-like receptors (TLR) play a key role in the recognition of pathogenic organisms. Fibronectin, an extracellular matrix protein, is considered a potent stimulator of the innate immune system through TLR4. In bacterial meningitis, several extracellular matrix proteins and bacterial compounds are elevated in the CSF. For this reason, we hypothesized that these molecules may jointly stimulate the innate immune system and increase neuronal damage in bacterial meningitis. Concentrations of fibronectin were elevated in the CSF of patients suffering from bacterial meningitis, but not in patients with multiple sclerosis, when compared with control patients without CSF abnormalities. In primary cultures of mouse microglial cells, co-administration of fibronectin at concentrations occurring in the CSF in bacterial meningitis (10 microg/mL) with defined TLR agonists [lipopolysaccharide (TLR4), the synthetic lipopeptide tripalmytoyl-cysteinyl-seryl-(lysyl)3-lysine (TLR2) and single-stranded unmethylated cytosine-guanosine oligodesoxynucleotide (TLR9)] led to an additive release of nitric oxide and tumor necrosis factor-alpha when compared with the release elicited by either compound alone. In conclusion, the inflammatory reaction to bacterial compounds can be aggravated by endogenous fibronectin at elevated levels during bacterial CNS infections. This additive or synergistic effect may contribute to neuronal damage during bacterial meningitis.