To identify proteins which bind to mouse wee1 kinase, the yeast "two-hybrid" system was used with a mouse cDNA library. Using the carboxyl half of weel kinase, the 14-3-3 zeta protein was isolated. Recombinant 14-3-3 zeta was demonstrated to bind to wee1 kinase in vitro. The wee1 kinase phosphorylated by cdc2 kinase also bound to 14-3-3 zeta protein. When both wee1 kinase and 14-3-3 zeta were transfected into COS-1 cells, they formed a complex in a cell. The sequence of wee1 kinase necessary for the binding was tested by a two hybrid system expressing different lengths of peptides derived from wee1 kinase. Both the entire kinase domain and a sequence in the carboxyl terminus was thought to be necessary for the binding. The function of 14-3-3 zeta protein remained to be elucidated in relation to the regulation of G2 to M phase transition through wee1 kinase.