In eukaryotic cells, the kinase activity of the mitosis-promoting complex composed of cyclin B and Cdc2 (Cdk1) is negatively regulated by the phosphorylation of Cdk1 on threonine or tyrosine residues within its ATP binding domain. We identified human Wee1B by searching a sequence database. The predicted human Wee1B protein comprises 561 amino acids. Northern blot analysis revealed that human Wee1B mRNA is particularly abundant in testis. Interestingly, RT-PCR using early embryos revealed that the Wee1B product was readily detectable at the mature oocyte, but abruptly disappeared at embryonic day 2.5, suggesting that the amount of Wee1B mRNA is dependent on the maternal expression. GFP-Wee1B showed a predominantly nuclear localization in HeLa cells. Human Wee1B was able to rescue the lethal phenotype of the fission yeast wee1-50Deltamik1 mutant, and over-expression of the human protein in these cells resulted in cell elongation as a result of arrest of the cell cycle at the G2-M transition. Recombinant Wee1B effectively phosphorylated cyclin B-associated Cdk1 on tyrosine-15, resulting in an inactivation of the kinase activity of Cdk1. We identified human Wee1B as a novel Cdk1-inhibitory kinase. The identification of this new member of the Wee1 family suggests that inhibition of Cdk1 is mediated at multiple levels in mammals.