In mammalian cells, p34cdc2 kinase undergoes phosphorylation at threonine-14, tyrosine-15 and threonine-161 in the S and G2 phases of the cell cycle. At the onset of mitosis, the kinase becomes dephosphorylated at threonine-14 and tyrosine-15, resulting in activation. Cdc25 phosphatase has been shown to dephosphorylate tyrosine-15 in vitro, but whether it also does at threonine-14 remains unclear. In this study, we have found that human cdc25B phosphatase dephosphorylates both threonine-14 and tyrosine-15 but not threonine-161.