The kinetic parameters of Cex, a family 10 xylanase from Cellulomonas fimi, were determined at various pH levels using soluble birchwood xylan (BWX) as a natural polymeric substrate along with three other synthetic aryl-β-D-xylobioside substrates. Using BWX, a high level of substrate inhibition was observed which increased with decreasing pH. In contrast, typical Michaelis-Menten-type profiles were obtained using the three aryl-β-D-xylobiosides as substrates. The kcat values determined using o-nitrophenyl-β-D-xylobioside did not change as the pH increased, whereas the kcat values obtained with BWX, phenyl-β-D-xylobioside and p-nitrophenyl-β-D-xylobioside decreased, suggesting that the presence of an ortho nitro group affects the activity displayed by Cex. These differences were not observed with XynB from Clostridium stercorarium F9, a member of the same family of xylanases as Cex. These results indicate that a careful evaluation is required when employing substituted aryl-β-D-xylobiosides in the characterization of xylanases.