Marek's disease (MD) is a lymphoproliferative disease of chickens caused by cell-associated Marek's disease virus (MDV). The Us3 protein kinase expressed by MDV has been shown to be involved in various stages of the viral life cycle. Deletion of the Us3 open reading frame resulted in an accumulation of primarily enveloped virions in the perinuclear space which led to a reduction in viral titers (Schumacher et al., 2005). It was also shown that the Us3 protein is involved in actin stress fiber breakdown. In this study we constructed a recombinant virus (Us3220A) to determine the significance of the Us3 kinase activity. Disruption of the kinase activity was achieved by substituting a lysine in the active site of the protein with an alanine. Titers of the kinase-negative virus were reduced when compared to parental virus in a similar fashion as the previously described Us3 deletion mutant. We were able to show complete absence of direct phosphorylation of an MDV-specific phosphoprotein, pp38 by pUs3220A. Surprisingly, expression of pUs3220A mediated breakdown of the actin cytoskeleton 24h after transfection of chicken embryo fibroblasts in the same way wildtype pUs3 does. At 48h post transfection the actin cytoskeleton was fully restored in almost all transfected cells. Our results show that the Us3 and pp38 proteins comprise a kinase-substrate pair but that not all of Us3 functions are mediated by its kinase activity.