PREPARATION of mitochondria from Neurospora usually requires quite drastic treatments such as micro-mills, glass beads, sand and disruption of protoplasts. It also requires from 2 to 12 h after collection to isolate the mitochondrial fraction. We have developed a method for preparing mitochondria in 30 min using a combination of three new techniques—a nylon bag, a glass homogenizer and rapid centrifugation.