In general, systemic bacterial infections induce sickness behavior. In mice, lipopolysaccharide (LPS), a component of gram-negative bacteria, strongly reduces physical activity via toll-like receptor (TLR) 4. However, gram-negative bacteria, such as Salmonella, also express flagella containing flagellin (FG) which binds to TLR5 and induces pro-inflammatory cytokine production. It is unclear whether FG induces sickness behavior. To determine whether Salmonella administration regulates the reduction of voluntary physical activity in mice, male C3H/HeN (wild type) and C3H/HeJ (tlr4 gene mutated) mice were administered living Salmonella (live) and examined for wheel-running activity. The production of TNF-alpha in RAW 264 cells was measured by the ELISA assay under both live and heat-killed (HK) Salmonella conditions in vitro. Wheel-running activity in both C3H/HeJ and C3H/HeN mice after i.p. injection of live Salmonella (1 x 10(6) CFU/kg) was significantly lower than that in vehicle groups (p < 0.01, respectively), although wheel-running activity in C3H/HeJ mice was not reduced after i.p. injection of HK Salmonella (1 x 10(6) CFU/kg). Furthermore, TNF-alpha production from RAW 264 cells with HK Salmonella treatment at the early phase was higher than that with live Salmonella treatment. Interestingly, gentamicin-treated (GMT) Salmonella, (which have bacterial flagella removed), did not induce reduction of wheel-running activity, although injection of the flagella-rich supernatant of GMT Salmonella significantly reduced it (p < 0.01). Indeed, FG treatment also induced reduction of wheel-running activity in mice (p < 0.01). Our findings suggest that the Salmonella-induced reduction of voluntary physical activity might be regulated by FG via TLR5, but not LPS via TLR4 in mice.