Two procedures are described for isolating free (nonsynaptosomal) mitochondria from rat brain. Both procedures employ a discontinuous Percoll gradient and yield well coupled mitochondria which exhibit high rates of respiratory activity and contain little residual contamination by synaptosomes or myelin. The procedures are considerably more rapid than methods described previously for the isolation of brain mitochondria and do not require an ultracentrifuge or swing-out rotor. The first method separates mitochondria by gradient centrifugation from a P2 (crude mitochondrial) fraction and is likely to be widely applicable for studies in which at least 500 mg of tissue are available as starting material. In the second method, the unfractionated homogenate is subjected directly to gradient centrifugation. This method requires the preparation of more gradients (per gram of tissue) than the first method and yields a subcellular fraction with slightly more synaptosomal contamination. However, this second procedure is more rapid, requires less manipulation of the tissue, and is suitable for obtaining mitochondria with well preserved metabolic characteristics from subregions of single rat brains.