The small molecule salubrinal has antiviral activity against herpes simplex virus-1 (HSV-1) and inhibits dephosphorylation of eIF2 alpha mediated by the HSV-1 protein ICP34.5. We investigated whether salubrinal's activities in infected cells depend on ICP34.5. An ICP34.5 deletion mutant was as sensitive as wild type HSV-1 to salubrinal inhibition of plaque formation in Vero cells. However, salubrinal induced formation of syncytia in infected Vero cells, which was enhanced by ICP34.5 mutations. Expression of HSV-1 US11 with immediate early kinetics, which is known to suppress the effects of ICP34.5 mutations, resulted in slight resistance to salubrinal in murine embryonic fibroblasts, and substantial resistance in those cells when ICP34.5 was additionally mutated. ICP34.5 mutations, but not immediate early expression of US11, prevented salubrinal's ability to increase phosphorylation of eIF2 alpha during HSV-1 infection of Vero cells. Taken together, our data indicate that salubrinal has both ICP34.5-dependent and -independent activities in HSV-1 infected cells.