To investigate the substrate recognition at the minus subsites of glycoside hydrolase family 10 xylanases, the kinetic parameters of four xylanases on all four p -nitrophenyl β-glycosides of β-1,4-gluco/xylo-disaccharides were determined. All four xylanases hydrolyzed all the four substrates examined. The K _m values of all the enzymes on the four substrates lined up in the same order, indicating that both the subsites -1 and -2 of all the enzymes prefer xylose to glucose. The comparison of the parameters on the substrates gave detailed information on the substrate recognition at each subsite -1 and -2.