Vaccinia virus encodes two regulators of the cellular antiviral response. The E3L gene is thought to act primarily by sequestering double-stranded RNA, whereas the K3L gene is thought to act as a competitive inhibitor of the double-stranded RNA-dependent protein kinase, PKR. The broad host range associated with vaccinia virus replication appears to be related to the presence of these genes. The E3L gene is required for replication in HeLa cells, but is not required for replication in BHK cells. On the contrary, the K3L gene is required for replication in BHK cells, but is dispensable for replication in HeLa cells. Our results suggest that these cell lines varied in the expression of endogenous activatable PKR and that replication of vaccinia virus in different cell lines led to altered levels of double-stranded RNA synthesis from the virus. Vaccinia virus was able to overcome these cellular variations by regulating PKR activity through the synthesis of either E3L or K3L. The results suggest that vaccinia virus has evolved a broad host range by maintaining both the E3L and the K3L genes.