Neurospora crassa synthesizes and secretes an extracellular protease into its growth medium when an exogenous protein-serves as its principal source of sulfur, nitrogen, or carbon. The enzymes produced under these three growth conditions have been compared by a number of criteria. The results indicate that the same extracellular protease with a molecular weight of 31,000 is synthesized during the three different metabolic conditions. A regulatory mutant, which lacks a positive signal required for the synthesis of a family of related enzymes for sulfur metabolism, cannot synthesize the protease in response to a limitation for sulfur; yet, this same mutant is capable of producing the enzyme when it is limited for either nitrogen or carbon. A second regulatory mutant, defective in the control of nitrogen metabolism, fails to synthesize the protease only when it is limited for nitrogen. The evidence suggests that a single structural gene for this extracellular protease exists and that it is regulated in a complex fashion such that control signals arising from any one of the three distinct regulatory circuits can activate it for expression. A model is proposed for complex regulation of the synthesis of this enzyme.The eukaryotic fungus, Neurospora crassa, is a useful experimental organism with which to study regulation in higher organisms, and it has been increasingly used in this capacity in recent years (1-7). When Neurospora is grown in a medium in which an exogenous protein serves as the major or sole sulfur source, it synthesizes and secretes a protease into the medium (8). Similarly, an extracellular protease is produced upon limitation of either nitrogen (9)