The echinocandin derivative caspofungin (MK-0991, L-743,872) inhibits 1,3-beta-d-glucan synthesis and is active against several medically important fungi but is relatively ineffective against Cryptococcus neoformans. To investigate the mechanism of C. neoformans resistance, the prevalence of 1,3- and 1,6-beta-d-glucan linkages was determined in cells grown with and without caspofungin, using affinity-purified antisera and gold particle immunoelectron microscopy. Cryptococcal strains ATCC 24067 (serotype D) and MY2061 (serotype A) were studied. Growth at 4 microg/mL of caspofungin reduced both glucan linkages in both strains. However, growth at 2 microg/mL resulted in reduced 1,6-beta-d-glucan linkage only for MY2061. Inhibition of 1,6-beta-d-glucan synthesis may be an additional mechanism of action for pneumocandins. The relatively low efficacy of caspofungin against C. neoformans may result from reduced activity against C. neoformans glucan synthase or from yet undiscovered mechanisms of action operative in other fungal pathogens but not in C. neoformans.