A membrane-DNA complex has been isolated from Sarkosyl lysed mitochondria of Paramecium aurelia. Apart from DNA, which has been demonstrated to be of mitochondrial origin, the complex contains proteins involved in the process of DNA replication. When used as a sole source of enzymes and DNA template, the membrane-DNA complex was capable of synthesizing mitochondrial DNA in vitro. The incorporation of (3H)dTTP was dependent on the presence of endogenous mitochondrial DNA template, magnesium ions and required four deoxyribonucleoside triphosphates for its maximal activity. Actinonycin and ethidium bromide were found to have an inhibitory effect on the incorporation of the label. It was demonstrated that the in vitro DNA synthesis by the membrane-DNA complex involved formation of known replicative intermediates (lariats and dimers) characteristic of the replication of mitochondrial DNA in vivo. It was concluded, therefore, that the membrane-DNA complex represents a mitochondrial replication complex. The complex was further characterized in terms of protein content by SDS polyacrylamide gel electrophoresis. The electrophoretic comparison of complexes isolated from normally grown cells and cells exposed to drugs affecting mitochondrial DNA replication in Paramecium showed some protein variation. Preliminary attempts were made to remove the proteins not involved in the process of DNA replication by salt extraction. Electron microscope studies of the replication complex revealed a novel chromatin-like structure of the mitochondrial genome. Biochemical studies of the putative mitochondrial chromatin showed the presence of five basic proteins which behaved in terms of their acid solubility and electrophoretic mobility similarly to histones extracted from nuclear chromatin. Evidence was obtained for protection of mitochondrial DNA from random digestion by nucleases, thus implying the direct association of DNA with basic proteins. The evolutionary implications of the discovery of chromatin-like structure in mitochondria are discussed.