Exposure of HL-60 promyelocytes to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased incorporation of 32P into a Mr approximately 33,000 protein (NP33) found in the nuclear matrices prepared by treating cells with Triton X-100, nucleases, and 2 M NaCl (D. E. Macfarlane, J. Biol. Chem., 261: 6947-6953, 1986). We now report that 12-O-tetradecanoylphorbol-13-acetate causes phosphorylation of NP33 in U937, K562, HEL, Molt-3, and Raji cell lines, all of which are rapidly proliferating cells of hematopoietic origin. 12-O-Tetradecanoylphorbol-13-acetate caused a lesser degree of NP33 phosphorylation in peripheral blood lymphocytes and monocytes and none in granulocytes or platelets. The incorporation of 32P into NP33 was complete in about 10 min, and it was prevented or reversed by staurosporin, indicating that NP33 is continuously phosphorylated and dephosphorylated. NP33 was purified to homogeneity from Triton X-100-washed nuclei or whole cells by extraction with H2SO4, acetone precipitation, and preparative two-dimensional gel electrophoresis. The amino-terminal amino acid sequence of NP33 appears to be the same as that of ribosomal S6 protein. NP33 appears to be S6 protein copurifying with the nuclear matrix.