A (1→3),(1→4)-β-glucan synthase catalysing the synthesis of (1→3),(1→4)-β-glucan (mixed-linkage glucan) was investigated using microsomal membranes prepared from developing barley (Hordeum vulgare L. cv. Shikokuhadaka 97) endosperms harvested 21 days after flowering. The microsomal fraction produced (1→3),(1→4)-β-glucan by incorporation of [14C]Glc from UDP-[14C]Glc. The production of (1→3),(1→4)-β-glucan was ascertained by specific enzymatic digestion with endo-(1→3),(1→4)-β-glucanase (lichenase; EC 3.2.1.73) from Bacillus amyloliquefaciens, which released a radiolabelled trisaccharide (3-O-β-cellobiosyl-glucose) and a tetrasaccharide (3-O-β-cellotriosyl-glucose), the diagnostic oligosaccharides for the identification of (1→3),(1→4)-β-glucan. Digestion of the products with exo-(1→3)-β-glucanase (EC 3.2.1.58) from Basidiomycete QM806 released radiolabelled Glc, indicating that not only (1→3),(1→4)-β-glucans but also (1→3)-β-glucans (callose) had been formed due to the presence of (1→3)-β-glucan (callose) synthase (EC 2.4.1.34) in the microsomal fraction. The activity of (1→3),(1→4)-β-glucan synthase was maximal at pH 9.0 and at 25°C and in the presence of at least 2 mM Mg2+. The apparent Km and Vmax values for UDP-Glc were 0.33 mM and 480 pmol min−1 mg protein−1, respectively. Investigating the dependence of enzyme activity on developmental stage (7–35 days after flowering) of the endosperms, we found an increase of activity during the initial development reaching a maximum at 19 days, followed by a gradual decrease as the endosperms matured. The amount of (1→3),(1→4)-β-glucan in the cell walls of the endosperms, however, increased gradually towards maturation, even after 19 days. Analysing the relationship between enzyme activity and (1→3),(1→4)-β-glucan deposition in cell walls of endosperms prepared from 12 different barley varieties harvested 11–22 days after flowering showed that some varieties had both low activity and low glucan content, and in some both were high. But for several other varieties, the availability of donor substrate and other factors seem to influence the production of (1→3),(1→4)-β-glucan as well.