Particulate enzymes from suspension-cultured ryegrass (Lolium multiflorum Lam.) endosperm cells incorporated glucosyl residues from UDPglucose and GDP-glucose into f8-glucans. Three types of .8-glucans were produced from UDP-glucose: 1,3-,8-glucan; 1,4-/?-glucan; and mixed-linkage 1,3;1,4-.8-glucan. As in other systems, relatively more 1,4-.3-glucan was produced from a low (10 micromolar) UDP-glucose concentration, and relatively more 1,3-fi-glucan was produced from a high (1 millimolar) UDPglucose concentration. However, in ryegrass, 1,3;1,4-f8-glucan represented a major proportion of the products at both low and high UDP-glucose concentrations. The arrangement of linkages in the 1,3;1,4-fi-glucan was different at the two concentrations; at the low UDP-glucose concentration, more sequences of three consecutive 1,4-inkages were produced.The effects of pH, temperature, and metal ion concentrations on incorporation were dependent on the UDP-glucose concentration. At the low UDP-glucose concentration, incorporation into all three types of f8-glucan increased with increasing pH. At the high UDP-glucose concentration, 1,3-fi-glucan was the major product at pH 7 and below; 1,4-/3-glucan synthesis was optimal at pH 8; and synthesis of 1,3;1,4-,f-glucan was greatest above pH 8.With 10 micromolar GDP-glucose as substrate, 1,4-fi-glucan, but no 1,3;1,4-fi-glucan, was produced. Incorporation from either UDP-glucose or GDP-glucose was not influenced by the presence of the other.Cell-free preparations from higher plant sources incorporate glucosyl residues from nucleoside diphosphate glucose into /3-glucans containing 1,3-and 1,4-linkages. Some preparations produce both 1,3-,B-glucan and 1,4-,B-glucan, while other also produce mixed-linkage 1,3;1,4-,8-glucan (23). Evidence for the synthesis of these mixed-linkage f3-glucans has come from identification of 1,3; 1,4-,8-oligoglucosides in enzymic hydrolysates of the products formed by oat coleoptile (Avena sativa) (17) and mung bean hypocotyl (Phaseolus aureus) (15) preparations, from Smith degradation studies of the products from wheat root preparations (18), and from the susceptibility of 8-glucans produced by preparations from ryegrass (Lolium multiflorum) endosperm (7, 23) to hydrolysis by the 1,3; 1,4-13-glucan hydrolase from Bacillus subtilis (1).13-Glucans produced in vitro from ,IM concentrations of UDPglucose are rich in 1,4-,8-glucan linkages, but those formed from mm concentrations of UDP-glucose, by the same preparations, contain mostly 1,3-fl-linkages (22,29). In systems that produce 1,3;1,4-,8-glucan