Replication-conditional viruses destroy tumors in a process referred to as viral oncolysis. An important prerequisite for this cancer therapy strategy is use of viruses that replicate preferentially in neoplastic cells. In this study the DF3/MUC1 promoter/enhancer sequence is used to regulate expression of γ134.5 to drive replication of a Herpes simplex virus 1 (HSV-1) mutant (DF3γ34.5) preferentially in DF3/MUC1-positive cells. HSV-1 γ134.5 functions to dephosphorylate elongation initiation factor 2α, which is an important step for robust HSV-1 replication. After DF3γ34.5 infection of cells, elongation initiation factor 2α phosphatase activity and viral replication were observed preferentially in DF3/MUC1-positive cells but not in DF3/MUC1-negative cells. Regulation of γ134.5 function results in preferential replication in cancer cells that express DF3/MUC1, restricted biodistribution in vivo, and less toxicity as assessed by LD50. Preferential replication of DF3γ34.5 was observed in DF3/MUC1-positive liver tumors after intravascular perfusion of human liver specimens. DF3γ34.5 was effective against carcinoma xenografts in nude mice. Regulation of γ134. 5 by the DF3/MUC1 promoter is a promising strategy for development of HSV-1 mutants for viral oncolysis.