Objective: To investigate use of transcriptional regulatory elements (promoters) for tumor-associated antigens to achieve HSV-1 replication preferentially in cells that overexpress the tumor-associated antigens. Summary Background Data: An important advantage of replicating viruses for cancer therapy is their ability to simultaneously destroy tumor cells by replication and release progeny virion to infect and destroy adjacent cancer cells. This strategy requires regulation of the viral life cycle to obtain robust replication in neoplastic cells and minimize replication in nonneoplastic cells. Methods: Promoters for the human carcinoembryonic antigen (CEA) and MUC1/DF3 tumor-associated antigens were characterized and cloned into HSV-1 mutants as heterologous promoters regulating expression of two different HSV-1 genes. Viral replication in tumor cells and cytotoxicity was quantified with in vitro assays. Antineoplastic efficacy was characterized in a flank tumor xenograft model. Results: Several CEA promoters were cloned and characterized using luciferase reporter assays. The most specific promoter was used to construct and isolate two different HSV-1 mutants in which critical genes are regulated by this promoter (ICP4 and γ134.5). Similarly, the promoter for the DF3/MUC1 tumor-associated antigen was cloned into a third HSV-1 mutant such that it regulates expression of γ134.5. Regulation of ICP4 expression by the CEA promoter during HSV-1 infection overly attenuates viral replication. Regulation of γ134.5 expression by either the CEA promoter or the MUC1/DF3 promoter during HSV-1 infection modulates viral replication, with preferential replication in cells that overexpress the corresponding tumor-associated antigen. A single intratumoral inoculation of an HSV-1 mutant with the MUC1/DF3 promoter regulating γ134.5 expression results in significant antineoplastic activity in MUC1-positive pancreatic carcinoma xenografts as compared to mock inoculation. Conclusions: Promoters for tumor-associated antigens may be incorporated into the HSV-1 genome to regulate HSV-1 replication. The choices of HSV-1 gene and tumor-associated promoter are important determinants of success of this strategy. Because of its preferential replication in MUC1-positive tumors, an HSV-1 mutant with the MUC1/DF3 promoter regulating γ134.5 expression will undergo further examination as a novel cancer therapy agent.