(1,3)-beta-D-Glucan synthase is a cell wall synthesis enzyme that is the target of cilofungin, an antifungal agent of the lipopeptide class. Cilofungin's glucan synthase inhibitory activity, MIC, and effective dose 50% in a systemic infection mouse model tend to correlate for Candida albicans. This correlation is not seen in Aspergillus fumigatus. MICs for cilofungin against A. fumigatus were consistently > 125 micrograms/ml while the effective dose 50% in a systemic aspergillosis model was determined to be 20.6 mg/kg. To begin to understand this discrepancy, we examined the A. fumigatus glucan synthase. This cell wall enzyme was prepared and its activity was measured by [14C]-glucose incorporation from UDP-[U-14C]glucose into an acid insoluble polymer formed in the presence of alpha-amylase. Enzyme activity in crude membrane preparations was measured in the presence of several antifungal agents. Enzyme inhibition results showed that 1 microgram/ml of papulacandin B, echinochandin B, aculeacin A and cilofungin all inhibited A. fumigatus glucan synthase activity (40-71%) while 1 microgram/ml of amphotericin B, fluconazole, ketoconazole and nikkomycin did not affect enzyme activity. A correlation was therefore established between the inhibitory effect of cilofungin on the A. fumigatus glucan synthase and the effective dose 50% obtained in a systemic aspergillosis mouse model.