A β‐(1→4)‐xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley (Hordeum vulgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5′‐diphosphoxylose (UDP‐Xyl) into exogenous β‐(1→4)‐xylooligosaccharides derivatized at their reducing ends with 2‐aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β‐(1→4)‐xylotriose (Xyl3‐PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl4−8‐PA. Methylation analysis and enzymatic digestions with β‐xylosidase (EC 3.2.1.37) and endo‐β‐(1→4)‐xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β‐(1→4)‐linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X‐100 and 5 mM MnCl2. The apparent Michaelis constant and maximal velocity of the enzyme for Xyl3‐PA were 2.1 mM and 25 400 pmol min−1 mg protein−1, respectively. The enzyme also transferred [14C]Xyl from UDP‐[14C]Xyl into higher β‐(1→4)‐xylooligosaccharides and birchwood xylans through β‐(1→4)‐linkages. The enzyme activity varied according to the stage of development (7–35 days after flowering) of the endosperms. Maximal activity occurred at 13–16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11–22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.