1. 1. Isolated chick-liver mitochondria were allowed to incorporate bromo[ 3H]-deoxyuridine for 30 min. The product DNA was purified and fractionated on a sucrose gradient. Fractions co-sedimenting with mitochondrial closed circular duplex DNA (I∗) and open circles (II∗) were analysed in neutral CsCl gradients before and after sonication. Intact bromouridine-labelled I∗ and II∗ were not detectably displaced in relation to marker mitochondrial DNA, but after sonication, fully hybrid DNA fragments were present. The hybrid sections represent less than 12 % of the I∗ molecule. 2. 2. When I∗, labelled with [ 3H]- or [ 14C]thymidine, is denatured by alkali or heating in the presence of formaldehyde, the bulk of the radioactive DNA was released as heterogeneous fragments with a sedimentation coefficient below 20 S and a peak around 10 S. The unlabelled DNA, in contrast, sedimented as intact supercoil DNA with some material in the region of intact single-stranded mitochondrial DNA. 3. 3. Thermal chromatography on hydroxyapatite showed that the radioactive DNA in I∗ starts to elute 14° below the elution of cold mitochondrial DNA; the difference in T m was 4°. 4. 4. These results and those presented in the accompanying paper indicate that I∗ consists of newly synthesized Okazaki fragments hydrogen-bonded to one or both strands of a mitochondrial closed circular duplex. I∗ may, therefore, represent a novel intermediate in the replication of DNA circles.