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Microsomal chitinase from yeast and hyphal cells of Candida albicans was activated endogenously by incubation at 30°C and exogenously by trypsin. The putative activating factor of yeast cells was separated from chitinase activity by fractionation of lysed protoplasts on an Iodixanol density gradient. The vacuole fraction contained no significant chitinase activity, but was enriched in chitinase activating factor. Activity of microsomal chitinase increased upon incubation with this, but no other gradient factor. Results suggest that the regulatory system governing microsomal chitinase activity, like that governing chitin synthase, involves a 'vacuolar' activating factor in Candida albicans.