Molecules of mitochondrial DNA (mtDNA) are packed into nucleic acid–protein complexes termed mitochondrial nucleoids (mt-nucleoids). In this study, we analysed mt-nucleoids of the yeastCandida parapsilosis, which harbours a linear form of the mitochondrial genome. To identify conserved as well as specific features of mt-nucleoids in this species, we employed two strategies for analysis of their components. First, we investigated the protein composition of mt-nucleoids isolated fromC. parapsilosismitochondria, determined N-terminal amino acid sequences of 14 proteins associated with the mt-nucleoids and identified corresponding genes. Next, we complemented the list of mt-nucleoid components with additional candidates identified in the complete genome sequence ofC. parapsilosisas homologues ofSaccharomyces cerevisiaemt-nucleoid proteins. Our approach revealed several known mt-nucleoid proteins as well as additional components that expand the repertoire of proteins associated with these cytological structures. In particular, we identified and purified the protein Gcf1, which is abundant in the mt-nucleoids and exhibits structural features in common with the mtDNA packaging protein Abf2 fromS. cerevisiae. We demonstrate that Gcf1p co-localizes with mtDNA, has DNA-binding activityin vitro, and is able to stabilize mtDNA in theS. cerevisiaeΔabf2mutant, all of which points to a role in the maintenance of theC. parapsilosismitochondrial genome. Importantly, in contrast to Abf2p,in silicoanalysis of Gcf1p predicted the presence of a coiled-coil domain and a single high-mobility group (HMG) box, suggesting that it represents a novel type of mitochondrial HMG protein.