Metabolic suicide gene transfer is widely applied for gene therapy of cancer, and retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene are commonly used in clinical trials. Most of these vectors contain positive selectable markers that undoubtedly facilitate the determination of viral titer and the identification of high-titer producer clones. However, the presence of additional transcriptional units may result in reduced expression of the gene of interest. The use of fusion genes expressing bifunctional proteins may help to overcome this problem. We have constructed a retroviral vector carrying the TNFUS69 chimeric gene, which originates from the fusion of the HSV-tk and neomycin phosphotransferase II genes, and evaluated the functional expression of the encoded fusion protein. In vitro, expression of the fusion gene conferred to target cells both resistance to neomycin and selective sensitivity to the antiherpetic drugs ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine. Cells transduced with the fusion gene, however, showed reduced ability to phosphorylate ganciclovir compared with cells expressing the native HSV-tk. Therefore, although the fusion gene may be used as a constituent of retroviral cassettes for positive and negative selection in vitro, its usefulness for suicide gene transfer applications in vivo may depend upon the possibility of using (E)-5-(2-bromovinyl)-2'-deoxyuridine in a clinical context.