A rapid method (about 1.5 h) for the isolation of intact functional mitochondria from neurons and astrocytes in primary culture is described. Mitochondria isolated by this method are metabolically active and tightly coupled as shown by respiratory control ratio values, which were about 4 with glutamate-malate as substrate. The activities of marker enzymes revealed the occurrence of a low degree of cytosolic (5%) or synaptosomal (5.5%) contamination in the mitochondrial fractions. In addition, the activity of citrate synthase was increased by 4 fold in both neuronal and astrocytic mitochondria with respect to values found in cell homogenates. These results confirm that the method affords mitochondrial preparations from cultured brain cells at suitable levels of purity and enrichment for the study of their mitochondrial function. Since mitochondrial damage has been associated with the pathogenesis of certain neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases (P. Chagnon, C. Betard, Y. Robitaille, A. Cholette, D. Gauvreau, Distribution of brain cytochrome oxidase activity in various neurodegenerative disease, Neuroreport 6 (1995) 711-715 [6]; S.J. Kish, C. Bergeron, A. Rajput, S. Dozic, F. Mastrogiacomo, L. Chang, J.M. Wilson, L.M. DiStefano, J.N. Nobrega, Brain cytochrome oxidase in Alzheimer's disease, J. Neurochem. 59 (1992) 776-779 [10]; A.H.V. Schapira, J.M. Cooper, D. Dexter, J.B. Clark, P. Jenner, C.D. Marsden, Mitochondrial complex I deficiency in Parkinson's disease, J. Neurochem. 54 (1990) 823-827 [15]), the method described here shed light on the possible susceptibility of neuronal or astrocytic mitochondria to deleterious effects of these diseases.