Kinetoplast DNA, the mitochondrial DNA in trypanosomatids, is a network of thousands of interlocked circles. Most of these circles are minicircles and a few are maxicircles. Minicircles replicate, after decatenation from the network, by a Cairns-type mechanism. The minicircle progeny then reattach to the network (Englund, P. T. (1979) J. Biol. Chem. 254, 4895-4900). We have now discovered a novel intermediate in Crithidia fasciculata minicircle replication. It is a highly gapped 2.5-kilobase free minicircle with nascent fragments of only 20 to 110 nucleotides. These fragments are nonligasable, and some remain nonligasable even after gap filling with DNA polymerase. Solution hybridization studies show that the nascent fragments are predominantly, if not exclusively, heavy strand.