Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically in controlled batch cultures. Of all factors investigated in a series of initial screening experiments, culture pH and nitrogen concentration in particular strongly affected extracellular protease activities. For instance, at a culture pH of 4, protease activity was higher than at pH 5, and protease activity increased with increasing concentrations of ammonium as nitrogen source. Interestingly, an interdependence was observed for several of the factors studied. These possible interaction effects were investigated further using a full factorial experimental design. Amongst others, the results showed a clear interaction effect between nitrogen source and nitrogen concentration. Based on the observed interactions, the selection of environmental factors to reduce protease activity is not straightforward, as unexpected antagonistic or synergistic effects occur. Furthermore, not only were the effects of the process parameters on maximum protease activity investigated, but five other protease-related phenotypes were studied as well, such as maximum specific protease activity and maximum protease productivity. There were significant differences in the effect of the environmental parameters on the various protease-related phenotypes. For instance, pH significantly affected final levels of protease activity, but not protease productivity. The results obtained in this study are important for the optimization of A. niger for protein production.