Vaccinia virus is being investigated as a replicating vector for tumor-directed gene therapy. However, the majority of cancer patients have preformed immunologic reactivity against vaccinia virus, as a result of smallpox vaccination, which may limit its use as a vector. The Yaba-like disease (YLD) virus was investigated here as an alternative, replicating poxvirus for cancer gene therapy. We have demonstrated that the YLD virus does not cross-react with vaccinia virus antibodies, and it replicates efficiently in human tumor cells. YLD virus can be expanded and purified to high titer in CV-1 cells under conditions utilized for vaccinia virus. The YLD virus RNA polymerase was able to express genes regulated by a synthetic promoter designed for use in orthopoxviruses. We sequenced the YLD virus TK gene and created a shuttle plasmid, which allowed the recombination of the green fluorescent protein (GFP) gene into the YLD virus. In a murine model of ovarian cancer, up to 38% of cells in the tumor expressed the GFP transgene 12 days after intraperitoneal virus delivery. YLD virus has favorable characteristics as a vector for cancer gene therapy, and this potential should be explored further.Poxviruses have unique characteristics which make them appealing as vectors for cancer gene therapy (4, 25). They have been investigated as vectors for delivery of tumor-associated antigens, cytokines, and costimulatory molecules in cancer patients, for the development of an antitumor immune response (5,17,22,32). Recently, laboratory experiments have supported the utility of vaccinia virus (VV) as a vector for tumordirected delivery of genes for enzyme-prodrug therapy and sensitization to systemic treatment with tumor necrosis factor (13,15,30). A replicating virus has distinct advantages over nonreplicating vectors for these tumor-directed applications, as it leads to an increase in the percentage of cells within a tumor that express the therapeutic gene over time (23,35). VV is an efficient, replicating virus that leads to high levels of transgene expression, selectively in tumor tissue when delivered systemically, and this can lead to a significant antitumor response. Selective mutations of the virus may enhance tumor specificity (29) Clinical trials with intravascular delivery of mutant VV will likely be hampered by the high percentage of cancer patients with preformed immunity against the virus as a result of vaccination against smallpox. High levels of circulating antibody titers and cytotoxic T cells recognizing VV can be detected many years after vaccination, and it is likely that this preformed immune reactivity will prevent adequate infection and spread of VV throughout a tumor when used as a vector for tumor-directed gene therapy. An alternative replicating poxvirus vector may mediate the selective, high transgene expression within tumors, without immune cross-reactivity. In general, the host range for poxviruses that do not cross-react with orthopoxviruses is quite limited, and although members of the avipoxvirus genu...