Glycogen synthase kinase 3 beta (GSK3 beta) is a Ser/Thr kinase that is involved in numerous cellular activities. GSK3 beta is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3 beta. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3 beta. In transfected CHO cells, it was demonstrated that PYK2 tyrosine phosphorylates GSK3 beta in situ. The two kinases also coimmunoprecipitated. Furthermore, GSK3 beta was tyrosine phosphorylated in vitro by an active, wild type PYK2, but not by the inactive, kinase dead form of PYK2. Therefore, this study is the first to demonstrate that GSK3 beta is a substrate of PYK2 both in vitro and in situ.