The nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting diacylglycerol (DG) into phosphatidic acid, terminate DG-dependent events. It has been demonstrated that nuclear DGK- interferes with cell cycle progression. We previously reported that nuclear DGK- expression increased during myogenic differentiation, whereas its down-regulation impaired differentiation. Here, we evaluated the possible involvement of nuclear DGK- in cell cycle progression of C2C12 myoblasts. Overexpression of a wild-type DGK-, which mainly localized to the nucleus (but not of a kinase dead mutant or of a mutant that did not enter the nucleus), blocked the cells in the G₁ phase of the cell cycle, as demonstrated by in situ analysis of biotinylated-16-dUTP incorporated into newly synthesized DNA and by flow cytometry. In contrast, down-regulation of endogenous DGK- by short interfering RNA (siRNA) increased the number of cells in both the S and G₂/M phases of the cell cycle. Cell cycle arrest of cells overexpressing wild-type DGK- was accompanied by decreased levels of retinoblastoma protein phosphorylated on Ser-807/811. Down-regulation of endogenous DGK-, using siRNA, prevented the cell cycle block characterizing C2C12 cell myogenic differentiation. Overall, our results identify nuclear DGK- as a key determinant of cell cycle progression and differentiation of C2C12 cells. --Evangelisti, C., Tazzari, P. L., Riccio, M., Fiume, R., Hozumi, Y., Falà, F., Goto, K., Manzoli, L., Cocco, L., Martelli, A. M. Nuclear diacylglycerol kinase- is a negative regulator of cell cycle progression in C2C12 mouse myoblasts.