A 26-kDa protein with highly basic pI was purified from the mitochondrial (mt-) nucleoids of the yeast Pichia jadinii by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 26-kDa protein has the ability to introduce a supercoil into circular plasmid DNA in the presence of topoisomerase I and to package mtDNA into nucleoid-like aggregates. The mt-nucleoids isolated from P. jadinii cells were disassembled in the presence of 2 M NaCl and reassembled into nucleoid-like aggregates by the removal of the salts. During the course of the reassembly of the mt-nucleoids, three specific proteins of 20 kDa, 26 kDa and 56 kDa predominantly precipitated after the centrifugation of the reassembled mt-nucleoids. These results suggest that the 26-kDa protein of P. jadinii has a similar function in the packaging of mtDNA to Abf2p, a major mitochondrial DNA-binding protein in Saccharomyces cerevisiae.