Mcm10 (Dna43) is an essential protein for chromosomal DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog that interacts with mammalian Orc2 and Mcm2-7 complex. We additionally demonstrated that human Mcm10 binds nuclease-resistant nuclear structures during S phase and dissociates from them in G2 phase. In this study, we further characterize the subcellular localization, modification, and expression levels of human Mcm10 protein throughout the cell cycle. Human Mcm10 protein decreased in late M phase, remained low during G1 phase, started to accumulate and bound chromatin at the onset of S phase. Proteasome inhibitors stabilized Mcm10 levels, suggesting that proteolysis is involved in the down-regulation of the protein in late M/G1 phase. Dissociation of Mcm10 from chromatin in G2/M phase was concomitant with alterations in the electrophoretic mobility of the protein. Treatment with l phosphatase revealed that mobility shifts were due to hyperphosphorylation. These results indicate that human Mcm10 is regulated by proteolysis and phosphorylation in a cell-cycle dependent manner. It is further suggested that mammalian Mcm10 is involved in S phase progression, and not the formation of a pre-replicative complex, as previously proposed from data on the S. cerevisiae protein.