Regulatory properties of a cellulose synthase (UDP-forming)(UDPglucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase, EC 18.104.22.168) have been demonstrated by using enzyme preparations derived from cells of Acetobacter xylinum. Preparation of a particulate fraction in the presence of 20% (wt/vol) polyethylene glycol-4000 (PEG-4000) yields enzyme with activity 3- to 10-fold higher than that previously reported. The enzyme prepared in this fashion also shows a further marked, specific activation by GTP. The Ka for GTP is 34 microM. Guanosine 5'-[gamma-thio]triphosphate, an analog of GTP, is even more effective than GTP (Ka for guanosine 5'-[gamma-thio]triphosphate = 17 microM). A large number of other nucleotides and nucleotide derivatives were tested with no effect. Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor necessary for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself has no synthase activity nor does it stimulate activity of the enzyme in the absence of GTP. In the presence of GTP, protein factor, and PEG-4000, initial rates of enzyme activity 200 times greater than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose.