Cyclin-dependent kinase (Cdk) belong to a family of Ser/Thr protein kinase is a representative cell cycle regulatory protein, which catalytic activity is tightly regulated by an interaction with cyclin. We identified and investigated a novel interaction between Cdk2 and cyclin O, a putative cyclin family protein containing cyclin-like domain that is conserved in cyclin family proteins. Co-immunoprecipitation using transiently transfected HEK293 cells showed that cyclin O interacted with Cdk2, especially with the active form of endogenous Cdk2. Cyclin O was expressed as several different bands between 45 and 50 kDa, due to post-translational modification. Interestingly, the co-expression of cyclin O and Cdk2 enhanced the intensity of upper band. The treatment of calf intestinal phosphatase (CIP) reduced the intensity of the uppermost band of cyclin O. Mass spectroscopic analysis showed that the 81st serine residue of cyclin O was phosphorylated by co-expression of Cdk2. The in vitro catalytic activity of Cdk2 phosphorylating histone H1 (HH1) was dramatically increased in cells over-expressing cyclin O. The Cdk2 kinase activity of cells over-expressing cyclin O S81A (mutant replaced 81st Ser to Ala) was less than that of cells over-expressing native cyclin O. Additional modifications of cyclin O were found in 16-51 amino acid residues. We currently investigate the effect of these modifications on the kinase activity of Cdk2. This work was supported by Basic Science Research Program through the National Research foundation (NRF) Funded by the Ministry of Education, Science and Technology of Korea (2011-0002725).