The interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (p68 kinase) has long been implicated as one of the antiviral agents responsible for overcoming virus infections. To investigate the antiviral potential of p68 kinase, we have generated a recombinant vaccinia virus that expresses human p68 kinase under the control of lac operator/repressor element. Upon induction of p68 kinase gene with the inducer isopropyl-beta-D-thiogalactoside (IPTG), we observed in cultured cells a severe (> 90%) inhibition of virus protein synthesis; this inhibition correlated with autophosphorylation of p68 kinase. As a result of inhibition in the synthesis of virus polypeptides, there was a 100-fold decrease in virus yields. When cells were infected with the recombinant virus expressing lys296-->arg296 mutant p68 kinase there was no reduction in virus yields. Our findings demonstrate that human p68 kinase once activated severely inhibits vaccinia virus replication as a result of inhibition of protein synthesis.