Alkaliphilic xylanases are not only important for their biotechnological applications, but also for their implications in protein structure-function. Here, we present for the first time the presence of single active site histidine residue in the microenvironment of GH-family 11 alkaliphilic xylanase from extremophilic fungus Penicillium citrinum MTCC 6489 using chemical modifications. The kinetic studies showed a time dependent inactivation of xylanase by OPTA or DEPC resulting in a pseudo-first-order kinetics with a second-order rate constant of 49.8 and 5.08 min-1 M-1, respectively. The difference spectrum of DEPC modified versus native protein exhibit an absorbance maximum at 244 nm characteristic of the formation of N-carbethoxyhistidine, which is completely reversed by neutralized hydroxylamine implying the presence of histidine residue. Moreover, the rate of inactivation shows pH dependence with an inflection point at 6.2. CD studies reveal no significant change in the DEPC modified xylanase conformation. Substrate dependent protection (0.5% xylan) from DEPC inactivation phenomenon conclusively proves the presence of histidine residue in the active site. To explore the presence of tryptophan in the active site xylanase is modified with NBS, which reveals its position in close proximity to active site, but not involved in catalysis. © 2007 Elsevier Inc. All rights reserved.