A major protein kinase independent of Ca2+, cyclic nucleotide or diacylglycerol, the activity of which becomes maximal when cells enter M phase, decreases at ana–telophase, and is low during interphase, has been purified to near homogeneity from starfish oocytes and its catalytic subunit identified as p34cdc2. M phase-promoting factor (MPF) was found to co-purify with the M phase-specific kinase throughout its purification. p34cdc2 does not have to be associated with any specific protein for expression of H1 histone kinase or MPF activities. When p34cdc2 is phosphorylated its protein kinase activity is inhibited, preventing entry into M phase, but once p34cdc2 becomes dephosphorylated, its protein kinase activity increases and M phase is initiated. A second peak of MPF activity was separated from p34cdc2 in the ammonium sulfate fraction treated with ATP-γ-S. It induced p34cdc2 dephosphorylation and the concomitant stimulation of its kinase activity when injected in Xenopus or starfish oocytes.