The nuclear matrix is thought to play a fundamental role in the nuclear structure and functions relating to cell proliferation and regulation of gene activity. Phosphorylation of nuclear matrix proteins has been observed, and multiple protein kinases may be involved in this process. Casein kinase 2 is a ubiquitous messenger-independent serine/threonine protein kinase that has been implicated in cellular growth and proliferation. The presence of immunoreactive casein kinase 2 in purified nuclear matrix preparations was demonstrated in rat liver and prostate tissues by employing specific antibodies. The enzyme was active toward the specific decapeptide substrate Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu and exhibited other properties characteristic of casein kinase 2. Phosphorylation of nuclear matrix proteins catalyzed by intrinsic protein kinase activity in intact nuclei was also observed, and the results suggested that this reaction was at least in part catalyzed by casein kinase 2. The profile of nuclear matrix proteins labeled with 32Pi in situ in intact liver cells was similar to that observed in nuclear matrix proteins isolated from nuclei labeled with [gamma-32P]ATP in vitro. Association of casein kinase 2 with the nuclear matrix may be of significance in the post-translational modification of certain proteins in this fraction.