Tumour necrosis factor-alpha (TNF-alpha) is one of the most prominent inflammatory mediators playing a central role in starting off the inflammatory reactions of the innate immune system. We identified a TNF-alpha-inhibitory activity in the saliva and salivary gland extract (SGE) from partially fed Ixodes ricinus ticks. Using mouse and human TNF-alpha specific ELISA, we showed that tick saliva or SGE markedly reduced the level of detectable cytokine. Both saliva and SGE inhibited the cytotoxic effect of TNF-alpha in a bioassay. Elimination of the TNF-alpha-inhibitory activity in SGE by trypsin digestion demonstrated that the anti-TNF-alpha factor is a protein. Fast protein liquid chromatography fractionation of SGE showed one peak of TNF-alpha-inhibitory activity corresponding to a protein with estimated molecular mass 23 kDa. The likely mechanism of the inhibitory effect is a direct binding of the cytokine. The TNF-alpha-inhibitory molecule seems to play an important role in the anti-inflammatory effect of tick saliva at the tick feeding site, providing a gateway to the host for tick-borne pathogens.