Mitochondria from Phymatotrichum omnivorum were purified using isopycnic gradient centrifugation in density gradients of sucrose or Percoll. Mitochondrial fractions obtained from sucrose gradients were found to be severely contaminated by microbody protein as evidenced by high catalase activity in the mitochondrial fraction. In addition, attempts to resuspend sucrose gradient purified mitochondria into an isosmotic medium have been unsuccessful. Percoll gradient purification of mitochondrial preparations yielded isosmotic mitochondria resolved from microbodies. Washed mitochondria obtained from self generated Percoll gradients exhibit low catalase activity and maintain high succinate dehydrogenase activity which suggests that Percoll gradient purification of mitochondria from P. omnivorum represents a superior method for the preparation of mitochondria for use in fungal metabolism studies.