Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor tyrosine kinase expressed in many cell types and enriched in neurons. PYK2 is a cytoplasmic enzyme activated by increases in cytosolic free Ca(2+) through an unknown mechanism. We report that depolarization or electrical stimulation of hippocampal slices induced a rapid and transient nuclear accumulation of PYK2. Depolarization of cultured neurons or PC12 cells also triggered a Ca(2+)-dependent nuclear accumulation of PYK2, much more pronounced than that induced by blockade of nuclear export with leptomycin B. Src-family kinase activity, PYK2 autophosphorylation and kinase activity were not required for its nuclear translocation. Depolarization induced a slight decrease in PYK2 apparent molecular mass, compatible with a Ca(2+)-activated dephosphorylation. Pretreatment of PC12 cells with inhibitors of calcineurin (protein phosphatase 2B), cyclosporin A and FK506, prevented depolarization-induced nuclear translocation and tyrosine phosphorylation of PYK2. Transfection with dominant-negative and constitutively active calcineurin-A confirmed the role of calcineurin in the regulation of PYK2 tyrosine phosphorylation and nuclear accumulation. Our results show that depolarization independently induces nuclear translocation and tyrosine phosphorylation of PYK2, and that both responses require calcineurin activation. We suggest that PYK2 exerts some of its actions in the nucleus and that the effects of calcineurin inhibitors may involve PYK2 inhibition.