In order to identify factors responsible for production of multiple forms of glucoamylase (GA) by Aspergillus niger Bo-1, the fungus was cultured in both complex and defined media in pH-controlled batch fermenters and chemostats. At all Culture conditions three forms of GA were produced with molecular weights of approx. 91 (GAI), 73 (GAII), and 59 kDa (GAIII). Data from batch fermentations with constant pH 3.0 and 5.0 showed a uniform distribution of extracellular GA forms throughout the fermentations and independent of culture growth phases. Furthermore, steady-state data from chemostat cultivations at constant pH 3.0 and 5.0 showed a similar distribution of extracellular GA forms and established that the nitrogen concentration of the medium (C/N ratio) did not affect the distribution of multiple forms of GA. The extracellular acid protease activity was only moderate when the fungus was Cultivated in batch and continuous fermentations with a constant pH of 3.0 or 5.0, whereas acidification of both complex and defined batch Culture media after 20 11 of growth induced a significant secretion of acid protease(s). The protease(s) present in the complex medium catalysed modification of the extracellular profile of the multiple forms of GA by degradation of GAI to GAII, whereas no proteolytic processing of the GA profile was observed in the defined medium. In vitro experiments confirmed that the pH-induced modifications of the GA multiple-form profile were caused by proteolytic and not spontaneous degradation of the GA forms at low pH. It was concluded that the observed modifications of the extracellular profile of GA isoforms in A. niger Bo-1 are due to changes in pH and medium composition.