The involvement of dipeptidyl peptidase (DP) IV/CD26 (DPP IV/CD26) family members in the pathogenesis of Crohn's disease (CD), an autoimmune inflammatory condition of the gut, is effected mainly through proteolytic cleavage of immunomodulatory substrates and DPP IV/CD26's costimulatory function. DP8 and DP9 are proteases with diverse functions including cell interactions, apoptosis, and immune response but their localization remains to be clarified. We assessed the impact of DPP IV/CD26 deficiency (CD26(-/-)) on the expression profiles of DP8 and DP9 by qPCR and immunodetection as well as quantified DP8/9 enzyme activity in distinctive phases of a chemically-induced CD model in mice. CD26(-/-) did not affect colon DP8 mRNA expression, while the physiological concentration of DP8 protein is decreased in CD26(-/-) mice but rises in inflammation (P<0.05). On the other hand, DP9 mRNA level is significantly increased in CD26(-/-) mice in inflammation as well as healing with the DP9 concentration being almost twofold increased (P<0.05) in all experimental points in CD26(-/-) mice compared to wild-type indicating the expected up-regulation in CD26(-/-) conditions. Surprisingly, dominantly intracellular DP8 and DP9 were found in abundance in serum. DP8/9 activity is decreased in the inflamed colon, whereas its contribution to the overall serum DPP IV/CD26-like activity is negligible, suggesting the importance of their extra-enzymatic functions. To summarize, CD induction generated gene, protein and enzymatic changes of DP8 and DP9 so their involvement in inflammation development and/or healing process is implicated, especially in CD26(-/-), and the question of their subcellular localization should be revised