The myogenic transcription factor Pax3, a member of the paired class homeodomain family of transcription factors, plays an essential role in early skeletal muscle development. We previously demonstrated that Pax3 is phosphorylated at three specific residues (Ser201, Ser205, and Ser209) and that the pattern of phosphorylation at these sites changes throughout early myogenesis. Further, we demonstrated that the protein kinase CK2 phosphorylates Pax3 at Ser205 and that this phosphorylation event is required for the subsequent phosphorylation of Ser201 by GSK3 beta. However, the kinase that phosphorylates Pax3 at Ser209 has yet to be identified. In the present work we use standard purification methods and in vitro biochemical analyses to provide solid evidence identifying the protein kinase CK2 as phosphorylating Pax3 at Ser209. Further, we qualitatively demonstrate that the phosphorylation of Pax3 at Ser209 by CK2 is enhanced when Ser205 is previously phosphorylated. Taken together, our results allow us to propose a mechanism to describe the ordered phosphorylation of Pax3 throughout early myogenesis. (c) 2012 Elsevier Inc. All rights reserved.