There are certain proteins present in mitotic cells that are absent in interphase cells. These are called mitotic factors, and they seem to regulate the condensation of chromosomes and the entry of cells into mitosis. The major objective of this research is to isolate and characterize factors from mitotic HeLa cells using the Xenopus laevis oocyte system for biological assay and to study whether the activity of the mitotic factors is associated with their phosphorylation. Taking advantage of the DNA-binding nature of the mitotic factors, we achieved a 200-fold purification by DNA-cellulose affinity chromatography. SDS-PAGE of partially purified mitotic factors indicated the presence of five to seven polypeptides with molecular weights ranging from 40 to 150 kilodaltons. All these peptides were found to be phosphoproteins as revealed by 32P labelling and autoradiography. Our studies indicated a strong correlation between phosphorylation of nonhistone proteins (NHP) extractable with 0.2 M NaCl and the entry of cells into mitosis on one hand, and dephosphorylation of these NHP and exit from mitosis on the other. We also discovered that certain factors (proteins) present in G1 phase HeLa cells are antagonistic to mitotic factors and these are named inhibitors of mitotic factors (IMF). IMF are activated at M-G1 transition and are present throughout the G1 period. IMF can be activated by UV-irradiation of Go or mitotic cells. IMF seems to have a role in chromosome decondensation. Two out of the four hybridoma clones we raised against mitotic HeLa cells specifically react with a set of NHP, which get phosphorylated during G2-M transition and dephosphorylated during M-G1 transition. These data suggest that phosphorylation by phosphoprotein kinases may be involved in chromosome condensation and initiation of mitosis, whereas dephosphorylation of these phosphoproteins by phosphatases results in chromosome decondensation at the end of mitosis. (N)