We have demonstrated that attenuated mutants of herpes simplex virus (HSV) have therapeutic potential for malignant brain tumors. In this report, we tested a ribonucleotide reductase-deficient (RR_-) HSV mutant as an experimental treatment for malignant brain tumors. The HSV-RR^- mutant hrR3, containing an Escherichia coli lacZ gene insertion in the ICP6 gene that encodes the large subunit of RR, was used in this study. We examined the cytopathic effect of hrR3 (0.1 plaque-forming unit/cell) on the U-87MG human glioblastoma cell line in vitro. Only 0.2% of U-87 cells were alive 67 h postinfection. Drug sensitivity assays demonstrated that hrR3 is hypersensitive to the antiherpetic agent ganciclovir. For in vivo studies, 10 animals harboring U-87MG tumors were randomly divided and treated intraneoplastically with either 5 × 10⁶ plaque-forming units of hrR3 or medium alone. The viral treatment group showed significant inhibition of tumor growth (P < 0.01; one-sided Wilcoxon rank test). Expression of the lacZ gene in hrR3, visualized by 5-bromo-4-Chrolo-3-indolyl-β-d-galactopyranoside histochemistry, could be detected in treated tumors. The therapeutic potential of this HSV-RR^- mutant for malignant gliomas is discussed.