This is the final version of the article. It first appeared from Taylor & Francis via http://dx.doi.org/10.1080/09168451.2015.1046365. ; ?-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single ?-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In the present study, we examined the action of an endo-?-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley ?-1,3:1,4-glucan. To find the minor structure on which the endo-?-1,3(4)-glucanase acts, we prepared oligosaccharides from barley ?-1,3:1,4-glucan by endo-?-1,4-glucanase digestion followed by purification by gel-permeation and paper chromatography. The endo-?-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization-time-of flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as ?-Glc-1,3-?-Glc-1,4-?-Glc-1,3-?-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-?-1,3(4)-glucanase acts on the cellobiosyl units of barley ?-1,3:1,4-glucan in an endo-manner. ; This work was supported in part by a grant-in-aid for Scientific Research to T. Kotake [Grant-in-Aid for Scientific Research no. 25514001] from Japan Society of the Promotion of Science; Y. Tsumuraya and T. Kotake [Grant-in-Aid for Scientific Research no. 24114006] from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Supports were also provided by BBSRC Sustainable Bioenergy Centre: Cell wall sugars program to P. Dupree [grant number BB/G016240/1].