Quantity and distribution of transgene-expressing tumor cells are central issues in cancer gene therapy. These are critical for the efficiency of tumor killing and for the bystander effect. In an attempt to combine the advantages of a potent bioactivating "suicide" gene with a marker gene for living cells, cDNA encoding cytochrome CYP4B1 was fused to the green fluorescent protein (GFP) cDNA. The resulting chimeric fusion protein, 4B1EGFP, was expressed in rodent and human glioma cell lines in culture. The ability of this recombinant enzyme to destroy tumor cells by converting the prodrug 4-ipomeanol (4-IM) into alkylating metabolites was evaluated in comparison with the cytotoxicity of the native CYP4B1 enzyme. The most sensitive 4B1EGFP-expressing glioma cell clone had a LD50 of 0.75 microgram/ml for 4-IM, as compared to a 4-IM LD50 of 0.5 microgram/ml in glioma cells expressing the native CYP4B1. A strong bystander effect mediated by cell-to-cell contact was present in the 4B1EGFP clones, allowing for more than 50% bystander kill at a ratio of expressing to non-expressing cells of 1:100. A herpes-simplex amplicon (pHSVPrPUC delta Hind) was constructed with the 4B1EGFP fusion protein, and recombinant helper-free HSV particles were packaged in Vero cells. Fisher 344 rats were inoculated with 4 x 10(5) 9L tumor cells to produce epidural tumor. Recombinant HSV particles were injected into the tumor at a dose of 1 x 10(7) pfu. Tumor was resected in living anesthetized animals 24, 48, and 72 hours after virus injection, and cryostat sections were evaluated by fluorescent microscopy. HSV-mediated delivery of the fusion protein to tumor cells was successfully demonstrated. In conclusion, the chimeric fusion protein 4B1EGFP retains essentially all features of the native CYP4B1 enzyme, and, moreover, offers advantages in terms of gene transfer visualization, which may lead to improvement of gene transfer strategies.