The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using ¹⁸F- or ¹²⁴I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine ([¹⁸F]FHBG). [¹⁸F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [¹⁸F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.