The lack of activity against factor Xa is in clear contrast to the predominant factor Xa inhibitory activity observed in tick saliva. We observed minimal thrombin inhibitory activity in adult saliva collected from engorged adult ticks. Recently, a 23 kDa protein was identified from a nymphal salivary gland yeast display library that appears to inhibit the formation of thrombin by targeting the activated factor Xa complex that precedes thrombin formation. A study by Chmelar et al has also shown that Ixodes ricinus salivary protein IRS-2 inhibits thrombin activity, albeit, at very high concentrations. We partially purified the thrombin inhibitory activity from adult tick guts by liquid chromatography. LC-MS/MS of the peptides in the active peak revealed the presence of a protein derived from the ISCW003862 locus. We named this protein Ixophilin on the basis of its strong homology with the thrombin inhibitors Boophilin and Hemalin from Boophilus microplus and Haemaphysalis longicornis respectively. ClustalW2 alignment of the full-length sequences of Ixophilin, Boophilin and Hemalin revealed 50% identity and 27% similarity over the 142 amino-acid full-length Hemalin and Boophilin. Ixophilin showed homology with Kunitzdomain containing super family of putative serine protease inhibitors, and contained 2 Kunitz domains,-one spanning amino acid residues 21 through 75, and a second, spanning residues 95 through 140. Kunitz domains have traditionally served as a preferred scaffold for evolution of tick anticoagulants. Taken together, our observations suggest that Ixophilin is Cycloheximide responsible, at least in part, for the thrombin inhibitory activity found in the tick midgut. rIxophilin showed the ability to inhibit thrombin by about 10�C20%. The activity of rIxophilin was much lower than the approximately 90% thrombin inhibitory activity observed in the native gut protein fractions, possibly due to the absence of tick-specific post-translational modifications, or to incorrect folding of rIxophilin. Ixophilin-derived peptides were obtained from SDS-PAGE bands with apparent molecular weights of 14, 16, and 18 kDa following thrombin-affinity chromatography, suggesting that Ixophilin may be activated by proteolytic cleavage. Incubation of rIxophilin with extracts from salivary glands and from midguts failed to increase the activity of the recombinant protein. Despite our inability to generate a potent rIxophilin, the sequence homology of Ixophilin with two other proteins, Boophilin and Hemalin that have been shown conclusively to be thrombin inhibitors, garners support for a thrombin inhibitory role for Ixophilin in the tick gut. Temporal and spatial analysis of ixophilin BU 4061T expression showed that it was preferentially expressed in the adult and nymphal gut and was induced upon feeding, consistent with a potential role for Ixophilin in preventing the clotting of the blood meal in the gut. Ixophilin expression levels were 100�C200-fold higher in the nymphal gut compared to the adult gut. ixophilin expression levels in fed and unfed larval stages were comparable, and significantly lower than that in nymphal and adult guts, suggesting that Ixophilin was possibly not the predominant anticoagulant in the larval stage. Since ixophilin expression was higher in the nymphal gut, we assessed the role of Ixophilin in nymphal feeding. Further, expression levels of ixophilin were significantly increased in repleting ticks.