The VRK1 kinase is a novel Ser-Thr kinase in the human kinome that diverged from the casein kinase 1 branch. These kinases phosphorylate transcription factors related to stress responses, such as p53. In this report we have studied the phosphorylation of the transcription factor c-Jun in its N-terminal region. The VRK1 protein phosphorylates c-Jun with a Km of 0.4 muM, and is not inhibited by SP600125. VRK1 phosphorylates c-Jun in Ser63 and Ser73 in vitro, the same residues targeted by the N-terminal kinase of c-Jun (JNK). This phosphorylation induces the stabilization and accumulation of the c-Jun protein. VRK1 phosphorylates the endogenous c-Jun in Ser63. VRK1 activates c-Jun dependent transcription, which is dependent on phosphorylation of Ser63 and Ser73. The c-Jun with Ser63Ala and Ser73Ala substitutions is not transcriptionally active when cotransfected with VRK1. VRK1 interacts with c-Jun but not with JNK. The cotransfection of VRK1 and JNK has an additive effect on the transcriptional activation of c-Jun indicating that they can cooperate when both are at suboptimal dose; otherwise, maximum effect by one of them prevents the effect of the other. The VRK1-c-Jun connection represents a component of a new signaling pathway whose upstream elements remain to be identified.