Maturation/M-phase-promoting factor is the universal inducer of M-phase in eukaryotic cells. It is currently accepted that M-phase-promoting factor is identical to the kinase cyclin B-Cdk1. Here we show that cyclin B-Cdk1 and M-phase-promoting factor are not in fact synonymous. Instead, M-phase-promoting factor contains at least two essential components: cyclin B-Cdk1 and another kinase, Greatwall kinase. In the absence of Greatwall kinase, the M-phase-promoting factor is undetectable in oocyte cytoplasm even though cyclin B-Cdk1 is fully active, whereas M-phase-promoting factor activity is restored when Greatwall kinase is added back. Although the excess amount of cyclin B-Cdk1 alone, but not Greatwall kinase alone, can induce nuclear envelope breakdown, spindle assembly is abortive. Addition of Greatwall kinase greatly reduces the amount of cyclin B-Cdk1 required for nuclear envelope breakdown, resulting in formation of the spindle with aligned chromosomes. M-phase-promoting factor is thus a system consisting of one kinase (cyclin B-Cdk1) that directs mitotic entry and a second kinase (Greatwall kinase) that suppresses the protein phosphatase 2A-B55 which opposes cyclin B-Cdk1.