The subcellular localization of glucose oxidase (EC 1.1.3.4) in Aspergillus niger N400 (CBS 120.49) was investigated by (immuno)cytochemical methods. By these methods, the bulk of the enzyme was found to be localized in the cell wall. In addition, four different catalases (EC 1.11.1.6) were demonstrated by nondenaturing polyacrylamide gel electrophoresis of crude extracts of induced and noninduced cells. Comparison of both protoplast and mycelial extracts indicated that, of two constitutive catalases, one is located outside the cell membrane whereas the other is intracellular. Parallel with the induction of glucose oxidase, two other catalases are also induced, one located intracellularly and one located extracellularly. Furthermore, lactonase (EC 3.1.1.17) activity, catalyzing the hydrolysis of glucono-delta-lactone to gluconic acid, was found to be exclusively located outside the cell membrane, indicating that gluconate formation in A. niger occurs extracellularly.