This work provides a rapid method for isolation of intact functional mitochondria from neurons and astrocytes in primary culture. By using this method, it was found that the respiratory control ratio was 1.5-fold greater in neuronal than in astrocytic mitochondria using both NAD-linked (glutamate/malate) and FAD-linked (succinate) substrates. The difference observed in RCR values was due to the lower rate of respiration in state 4 found in neurons as compared to that found in astrocytes, because both cell types showed the same rate of respiration in state 3. The P/O ratio was also higher in neurons than in astrocytes. Our results suggest that the coupling between the mitochondrial respiratory chain and oxidative phosphorylation is stronger in neurons than in astrocytes. These results may be of relevance for the understanding of the differential susceptibility of brain cells to impairments of energy metabolism observed in certain neurodegenerative diseases.