The incorporation of (3H) thymidine into isolated rat liver mitochondria in the presence of 0--0.5 mM ATP was stimulated by a polyanion with an average molecular weight of 10 000 daltons (at 50--200 micrograms/ml concentration) and containing in a proportion of 1 : 2 : 3 methacrylate, maleate and styrene. The above concentrations of the polyanion inhibit the incorporation of (3H) thymidine in the presence of 2 mM ATP and have no substantial effect on (14C)isoleucine incorporation in vitro. The mtDNA synthesis stimulatory effect of the polyanion is independent of its inhibitory effect on adenylate translocase. The polyanion does not enhance the nucleotide transport across the inner membrane of the mitochondria and the changes of thymidine phosphorylating activity are not involved in the increased mtDNA synthesis. It is supposed that the polyanion attached to the inner membrane of the mitochondria alters the binding of mtDNA to the inner membrane of the mitochondria. Thus, the polyanion might be an effective tool in studying the functional significance of mtDNA-membrane association in mtDNA replication.