The transcription factors ATF-2 and c-Jun are important for transactivation of varicella-zoster virus (VZV) genes. c-Jun is activated by the c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase pathway that responds to stress and cytokines. To study the effects of VZV on this pathway, confluent human foreskin fibroblasts were infected with cell-associated VZV for 1 to 4 days. Immunoblots showed that phosphorylated JNK and c-Jun levels increased in VZV-infected cells, and kinase assays determined that phospho-JNK was active. Phospho-JNK was detected after 24 h, and levels rose steadily over 4 days in parallel with accumulation of VZV antigen. The two main activators of JNK are MKK4 and MKK7, and levels of their active, phosphorylated forms also increased. The competitive inhibitor of JNK, SP600125, caused a dose-dependent reduction in VZV yield (50% effective concentration, congruent with 8 microM). Specificity was verified by immunoblotting; phospho-c-Jun was eliminated by 18 microM SP600125 in VZV-infected cells. Immunofluorescent confocal microscopy showed that phospho-c-Jun and most of phospho-JNK were in the nuclei of VZV-infected cells; some phospho-JNK was in the cytoplasm. MKK4, MKK7, JNK, and phospho-JNK were detected by immunoblotting in purified preparations of VZV virions, but c-Jun was absent. JNK was located in the virion tegument, as determined by biochemical fractionation and immunogold transmission electron microscopy. Overall, these results demonstrate the importance of the JNK pathway for VZV replication and advance the idea that JNK is a useful drug target against VZV.