We have cloned a mouse wee1 kinase cDNA (mwee1). The clone is 2258 bp in length and its open reading frame corresponds to 646 amino acid residues. The molecular weight of this kinase is 87 kDa in SDS-PAGE, which is about 1.7-fold larger than the human p50wee1 kinase reported previously. In a cell cycle, the mouse wee1 kinase is phosphorylated at M-phase, and an in vitro study using a mitotic extract revealed that phosphorylation occurs in the N-terminal domain, which is absent from the human wee1 kinase, resulting in inactivation of the kinase activity. The N-terminal domain or entire molecule is extensively phosphorylated by cdc2-cyclin B kinase. Furthermore, the activity of the wee1 kinase was reduced by phosphorylation with the mitotic extract which contained cdc2-cyclin B kinase.