We recently demonstrated in vitro that a mutant HSV-TK (mutant 75) expressed from an adenovirus (AdCMV-TK75) radiosensitized rat RT2 glioma cells significantly better than wild type HSV-TK (AdCMV-TK) in combination with acyclovir (ACV). To examine whether a similar improvement could also be observed in vivo, we tested these viruses in a syngeneic rat glioma tumor model (RT2/Fischer 344). First, we demonstrate that treatment with AdCMV-TK and ACV significantly radiosensitizes implanted gliomas and roughly doubles the mean survival time to 37 days, compared to 20 days for control animals implanted with Adbetagal-transduced cells (P<.02). Second, it was important to first examine the effect of AdCMV-TK75 and ACV on survival without any irradiation. We found that AdCMV-TK75 appeared to sensitize gliomas more efficiently than AdCMV-TK, although this difference was not significant ( P= .19 ). Third, and most importantly, in combined HSV-TK, ACV and irradiation experiments, we demonstrate that AdCMV-TK75 is superior over AdCMV-TK and significantly (P<.005) prolonged the survival of treated animals. Our results suggest that AdCMV-TK75 is far more efficient than AdCMV-TK in radiosensitizing rat glioma when administered in combination with ACV.