CDC25 phosphatases play key roles in cell proliferation by activating cell cycle-specific cyclin-dependent kinases (CDKs). We identified four new splice variants in the amino-terminal regulatory region of human cdc25C and one in cdc25A. All variants except one retain an intact catalytic domain. Alternative splicing results in loss of phosphorylation sites for kinases like CDK and the calcium/calmodulin-dependent kinase II (CaMKII), which influence CDC25 activity and compartmental localization. In NT2 teratocarcinoma cells, induced for nerve cell differentiation, the smaller sized variant of cdc25C was upregulated. At the protein level both phosphorylation state and isoform distribution differed between cell lines and cell cycle phases.