(1,3)Beta-D-glucan synthase (EC 2.4.1.34. UDP-glucose: 1,3-beta-D-glucan 3-beta-glucosyltransferase) uses UDP-glucose as substrate and catalyzes the polymerization of glucose ([1,3]-beta-linkages) to form the major carbohydrate component of the fungal cell wall. We have optimized in vitro assay conditions for (1,3)beta-glucan synthase activity from Cryptococcus neoformans. Cells lysed in 50 mM Tris, pH 7.75, containing 20% glycerol, 2 mM NaF, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl(2), 0.1% protease and phosphatase inhibitor cocktails, and 60 microM GTPgammaS produced maximum specific activity in vitro. We tested in vitro C. neoformans (1,3)beta-glucan synthase activity against the (1,3)beta-glucan synthase inhibitors, caspofungin and cilofungin, and have determined that (1,3)beta-glucan synthase activity is very sensitive (apparent K(i) of 0.17 +/- 0.02 microM and 22 +/- 5.7 microM, respectively) to these echinocandins. Taken together with high MICs for C. neoformans (caspofungin MIC, 16 microg/ml; cilofungin MIC, 64 microg/ml), our results indicate that C. neoformans is resistant to caspofungin and cilofungin by a mechanism(s) unrelated to (1,3)beta-glucan synthase resistance.