Yeast 1,3-beta-D-glucan synthetase (EC 2.4.1.34) activity is modulated by endogenous factors obtained by the extraction of different subcellular fractions with hot water. Cell wall fractions were enriched in activators while supernatant fractions also contained appreciable amounts of inhibitors. The action of these compounds requires the presence of EDTA. Maximal activation by the stimulatory material was reached when assayed in sonicated enzymatic preparations that had been obtained by mechanical breakage of cells in water. The activating material derived from cell wall fractions contained a mixture of low molecular weight compounds. They were found to be different from GTP as deduced from their resistance to alkaline phosphatase and different elution profile in gel filtration. The supernatant material was also heterogeneous with regard to both activators and inhibitors. The combined effect of GTP and activating material derived from cell wall fractions was supraadditive. The polymers synthesized in the absence and in the presence of the endogenous activator were characterized as beta-1,3-glucans on the base of their resistance to periodate and susceptibility to beta-glucanases. However, the length of the radioactive chains was greater when synthesized in the presence of the activator. This was mediated by an increase in the Vmax of the synthetase.