This paper reports on the protocols for the spectrophotometric determination of the enzymatic activity, the electrophoretic pattern and the cytochemical assay of glucose-6-phosphate dehydrogenase (G6PD) in different areas of rat brain. For the spectrophotometric assay we used the method of Glock and McLean. Non-denaturing polyacrylamide gel electrophoresis was performed using 1.5 mm thick slab-gel, with a 7.5% acrylamide separating gel and a 4% acrylamide stacking gel. NADP (0.01 mM) was included at the cathode buffer. The cytochemical assay was performed on unfixed cryostat sections, by incubating the slides for 5 min at 37 degrees C in the staining medium. A combination of these techniques offers a means for the study of the effect of age, drugs, oxidizing or reducing agents on the G6PD activity in different brain areas.