Cyclin-dependent kinase 5 (Cdk5) is primarily associated with brain development but it is also implicated in lens and muscle differentiation. We found that Cdk5 is also expressed in mouse ovary, and explored the possibility that it plays a role in that tissue. We show by Western blotting and immunohistochemistry that the known Cdk5 activator, p35, is also present in the mouse ovary. Cdk5 and p35 were detected in oocytes at all stages of the follicle. While Cdk5 was present in the cytoplasm and nucleus of the oocyte, p35 was observed only in the cytoplasm. Both proteins were detected in the cytoplasm of luteinized cells in the corpus luteum. Immunoprecipitation and histone H1 kinase assays revealed that they form an ovarian complex with considerable kinase activity. Phosphorylation assays showed that several ovarian proteins are substrates for Cdk5/p35 in vitro. Together our findings suggest that p35-associated Cdk5 activity plays an important role in the ovary, where it may regulate cell differentiation and apoptosis as it does in the brain.