Archaeal cell division cycle protein 6 (Cdc6) homologues are thought to be involved in the initiation process of DNA replication. In the present study, a biochemical characterization of the two Cdc6 proteins from the archaeon Thermoplasma acidophilum has been performed. Both TaCdc6-1 and TaCdc6-2 behave as monomers in solution and both are abundantly expressed in vivo. Further, TaCdc6-1 shows strong ability to undergo autophosphorylation compared to TaCdc6-2 and the autophosphorylation activity is not affected by DNA or MCM.