ORF47, a serine/threonine protein kinase encoded by varicella-zoster virus (VZV), has often been compared to the ubiquitous cellular kinase, casein kinase II (CKII). However, no direct comparison of the two protein kinases has been carried out. Herein, we show that the ORF47 kinase was resistant to heparin, while CKII activity is profoundly inhibited by the acidic molecule in vitro. ORF47 required the presence of polyamines (aliphatic, positively-charged molecules) for in vitro activity. When polyamines were depleted from MeWo cells prior to VZV infection by pretreatment with D,L-alpha-difluoromethylornithine, VZV replication was reduced by 80%. Finally, the substrate specificity of the ORF47 kinase was defined using an in vitro assay. The ORF47 kinase phosphorylated maltose-binding protein, the mouse IgG2A heavy chain, the rabbit IgG heavy chain, casein, VZV ORF62, and VZV ORF63. The ORF47 kinase failed to phosphorylate an ORF62 truncation mutant, glutathione-S-transferase, or VZV gB. In contrast, CKII weakly phosphorylated VZV gB in vitro. By analyzing the sequences of these substrates, the minimal ORF47 consensus sequence was deduced to be the following motif: S/T-X-D/E-D/E, with a marked preference for additional acidic amino acids in the -1 and +1 position.