Technical Abstract: A novel xylanase from Trichoderma reesei Rut C30, named XYN IV, was purified from the cellulolytic system of the fungus. The enzyme was discovered on its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA3Xyl3), releasing the reducing-end xylose residue. XYN IV exhibited catalytic properties different from previously described endo-'-1,4-xylanases of this fungus, XYNI, XYNII, XYNIII, and the xylan-hydrolyzing EGI. XYN IV was able to degrade various '-1,4-xylans, but was inactive on ß-1,4-mannans and ß-1,4-glucans. It showed both exo-and endo-xylanase activity. Rhodymenan, a linear soluble ß-1,3-ß-1,4-xylan, served as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4 encoding XYN IV was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30 harbouring also glucanases and mannanases. The xyn4 gene was expressed slightly on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in Pichia pastoris expression system exhibited identical catalytic properties as the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it could be liberation of a xylose residue from the reducing end of branched oligosaccharides that are resistant toward ß-xylosidase and other types of endoxylanases. By its catalytic properties XYN IV differs from bacterial GH30 glucuronoxylanases that recognize MeGlcA substituents as substrate specificity determinants.