A new method for the rapid and quantitative fluorometric determination of callose is described. In suspension-cultured cells of Glycine max, synthesis of callose starts within 20 minutes of treatment with chitosan and parallels over hours the accumulation of 1,3-linked glucose in the wall. Poly-L-lysine also elicits callose synthesis. The effect of chitosan is enhanced by Polymyxin B at low concentrations; this antibiotic alone at higher concentrations can also induce callose synthesis. Callose synthesis is immediately stopped when external Ca2l is bound by ethylene glycolbis-(2-aminoethyl ether)-N,N'-tetraacetate or cation exchange beads, and partly recovers upon restoration of 15 micromolar Ca21.Callose synthesis is observed only when membrane perturbation causing electrolyte leakage from the cells is induced by one of the above treatments. It does not appear to be due to de novo synthesis or proteolytic activation of 1,3-#-D-glucan synthase. It is concluded that this Ca24-dependent enzyme is directly activated by the influx of Ca24 occurring concomitantly with the leakage of cell constituents. This suggestion is also discussed in conjunction with the chitosan-induced synthesis of phytoalexin in the same cells.