Phosphorus MRS was evaluated as a monitor of tumour therapeutic response to the herpes simplex virus thymidine kinase suicide gene therapy paradigm. In vivo ³¹P spectra were obtained from subcutaneous rat C6 gliomas constitutively expressing the HSVtk gene post treatment with ganciclovir (GCV, 15 mg/kg i.p., twice‐daily). Significant regression (p<0.1) of tumour volume was observed 10 days after beginning GCV administration. However, no changes in tumour pH or energy metabolites from pre‐treatment values were observed. High‐resolution ³¹P spectra of tumour extracts revealed a statistically significant reduction in the phosphocholine to phosphoethanolamine ratio six days post‐GCV administration. These results indicate that the HSVtk/GCV‐induced killing of tumours is not associated with corresponding changes in ³¹P MRS‐observable energy metabolites and pH. The observed reduction in the PE/PC ratio may provide a non‐invasive in vivo indicator of therapeutic efficacy.