Globin genes are normally expressed only in erythroid cell lineages. However, we found that the endogenous alpha-globin gene is activated following infection of human fibroblasts and HeLa cells with herpes simplex virus (HSV), leading to accumulation of correctly initiated transcripts driven by the alpha-globin promoter. The alpha1- and alpha2-globin genes were both induced, but expression of beta- or zeta-globin genes could not be detected. Experiments using HSV mutants showed that null mutations in the genes encoding the viral immediate-early proteins ICP4 and ICP22 reduced induction approximately 10-fold, while loss of ICP0 function had a smaller inhibitory effect. Transient transfection experiments showed that ICP0 and ICP4 are each sufficient to trigger detectable expression of the endogenous gene, while ICP22 had no detectable effect in this assay. ICP4 also strongly enhanced expression of transfected copies of the alpha2-globin gene. In contrast, the adenovirus E1a protein did not activate the endogenous gene and inhibited expression of the plasmid-borne alpha2-globin gene. Previous studies have led to the hypothesis that chromosomal alpha-globin genes are subject to chromatin-dependent repression mechanism that prevents expression in nonerythroid cells. Our data suggest that HSV ICP0 and ICP4 either break or bypass this cellular gene silencing mechanism.