Background Herpes simplex virus 1716 (HSV1716), a selectively replication competent mutant of HSV1, is under investigation as an oncolytic viral therapy in human malignant glioma. As with similar therapies, a technique for measurement of viral replication and distribution over time following virus administration is required. Imaging expression of the HSV-thymidine kinase (HSV-tk) gene offers an opportunity for non-invasive assessment of viral distribution in living subjects. This is the first study to explore the use of HSV-tk as a reporter gene and radiolabelled thymidine analogue 5-[I-123] iodo-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) uracil (I-123-FIAU) as a marker substrate to non-invasively monitor HSV1716 replication in humans during treatment of high-grade glioma. Methods I-123-FIAU brain SPECT imaging was undertaken in eight patients receiving intra-tumoural injection of HSV1716, before and after administration of the virus. Baseline images were acquired 3 days prior to virus administration and between 1 and 5 days following virus administration. Region of interest analysis was used to investigate whether there was an increase in I-123-FIAU concentration following virus administration due to HSV-tk expression. Result Increased I-123-FIAU accumulation due to HSV-tk expression was not detected in this study. The possible explanations for this finding are explored and design options for future studies are discussed.