The biological role of archaeal proteins, homologous to the eukaryal replication initiation factors of cell division control (Cdc6) and origin recognition complex (ORC1), has not yet been clearly established. The hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as Sso) possesses three Cdc6/ORC1-like factors, which are named Sso Cdc6-1, Cdc6-2 and Cdc6-3. This study is a report on the biochemical characterization of Sso Cdc6-1 and Cdc6-3. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions by gel filtration analyses. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubble-containing synthetic oligonucleotides. The study has also showed, with surface plasmon resonance measurements, that Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3. These findings may provide important clues needed to understand the biological role that is played by each of these three Cdc6 factors during the DNA replication initiation process in the S. solfataricus cells.