This paper describes the optimization of production of ascosteroside, a novel antifungal agent with an alpha-linked glycoside of a lanosterone-type triterpenoid structure. Glucose, sorbose and inositol were determined to be the best carbon sources for the production of ascosteroside. Temperature affected levels of ascosteroside, with production being highest at 16 degrees C with 1% glucose, and lowest at 32 degrees C. Dissolved oxygen levels were found to be critical in the production of ascosteroside in fermenter cultures. In order for production of ascosteroside to occur in fermenter cultures, the threshold level of dissolved oxygen was found to be above 26%.