A population of small covalently closed non-mitochondrial circular DNA molecules was isolated from the petite-negative yeast Schizosaccharomyces pombe. The mean length of these molecules, possessing the same density as nuclear DNA (1.695 g/cm3) is 1.95 +/- 0.18 micrometer. The presence of these minicircles in crude mitochondrial preparations indicates their tight association with mitochondrial particles. Their disappearance after DNase treatment of mitochondria demonstrates their extramitochondrial location.