The preparation of mammalian cells for entry into mitosis is related to a cascade of G2 phase phosphorylations of several nuclear proteins driven by mitosis-specific protein kinases. Using a monoclonal antibody we have identified previously in mammalian cells a 125K/pI6.5 protein, associated with the nuclear matrix, and markedly increased in mitotic cells, which was named ‘mitotin’. Here, we show by short-term [35S]methionine labeling of cell cycle synchronized cells that this protein is synthesized at comparable rates throughout interphase. However, upon cycloheximide block of protein synthesis mitotin labeled during S phase is rapidly degraded, while the degradation of mitotin labeled during late G2 phase is abolished, resulting in its net and marked increase. The accumulation of mitotin in premitotic and mitotic cells is related to its phosphorylation and the metabolic stability of its two phosphorylated forms. The metabolic stabilization and accumulation of a nuclear matrix protein upon phosphorylation suggests the operation of a novel mechanism among the complex events preparing the cell for mitosis.