Factors which affect the production, activity, and stability of the extracellular protease of Beauveria bassiana have been investigated. During growth in liquid culture B. bassiana produced one extracellular protease that released free amino-nitrogen from gelatin. Unbuffered cultures (pH 7.0) containing gelatin and glucose showed a drop in pH to 2.5 which was inhibitory to protease activity as measured by an Azocoll assay. Protease activity was maximum at pH 8.5. The protease was stable for 24 h between pH 5 and 12, but was unstable at pH values below 5. In cultures buffered to pH 7.0, containing gelatin, synthesis of B. bassiana protease is repressed in the presence of ammonium and glucose, glycerol, trehalose, or mannitol but not with maltose. Partial repression of extracellular protease was observed in cultures buffered to pH 7.0 containing gelatin and glucose. Protease was not produced in cultures containing glucose and/or ammonium without gelatin. These results suggest that B. bassiana synthesizes and secretes an extracellular protease into its growth medium when an exogenous protein (gelatin) serves as its principal source of nitrogen and carbon. The regulation of extracellular protease synthesis in B. bassiana is consistent with a multiple control model.