Abstract We examined the mechanism of synthesis in vitro of (1→3),(1→4)β-d-glucan (β-glucan), a growth-specific cell wall polysaccharide found in grasses and cereals. β-Glucan is composed primarily of cellotriosyl and cellotetraosyl units linked by single (1→3)β-linkages. The ratio of cellotriosyl and cellotetraosyl units in the native polymer is strictly controlled at between 2 and 3 in all grasses, whereas the ratios of these units in β-glucan formed in vitro vary from 1.5 with 5 μm UDP-glucose (Glc) to over 11 with 30 mmsubstrate. These results support a model in which three sites of glycosyl transfer occur within the synthase complex to produce the cellobiosyl-(1→3)-d-glucosyl units. We propose that failure to fill one of the sites results in the iterative addition of one or more cellobiosyl units to produce the longer cellodextrin units in the polymer. Variations in the UDP-Glc concentration in excised maize (Zea mays) coleoptiles did not result in wide variations in the ratios of cellotriosyl and cellotetraosyl units in β-glucan synthesized in vivo, indicating that other factors control delivery of UDP-Glc to the synthase. In maize sucrose synthase is enriched in Golgi membranes and plasma membranes and may be involved in the control of substrate delivery to β-glucan synthase and cellulose synthase.