A sequential extraction procedure has been used to prepare three protein fractions from control and mitogen-stimulated [32P]-PO4-labelled fibroblasts, the first containing soluble and membrane-bound proteins, the second mainly the cytoskeletal proteins vimentin and actin, and the third, a chromatin-derived fraction enriched in histones. We describe here an analysis of the mitogen-stimulated changes in the [32P] labelled phosphoprotein composition of these fractions. The most obvious changes in response to epidermal growth factor, embryonal carcinoma-derived growth factor, basic fibroblast growth factor and bombesin were the rapid appearance of 33 and 15 kDa phosphoproteins in the chromatin-derived fraction. The epidermal growth factor- and basic fibroblast growth factor-stimulated 33 kDa phosphoprotein produced similar chymotryptic peptides and was phosphorylated on serine residues. DNAse/RNAse treatment of the lysates was essential for the extraction of the 33 kDa phosphoprotein. Further, its presence could be demonstrated in preparations of conventionally purified nuclei. An in situ extraction procedure has been used to provide morphological verification of the sequential extraction data. The final structure containing these phosphoproteins is clearly derived from nuclei, enriched in histones, stains for DNA and appears by electron microscopy, to be homogenously composed of chromatin-like material. Thus, we describe here the rapid mitogen-induced appearance of novel phosphoproteins in the nucleus, raising the possibility that they may be involved in orchestrating early nuclear responses to polypeptide growth factors.