Purpose Proton therapy precisely delivers radiation to cancers to cause damaging strand breaks to cellular DNA, kill malignant cells, and stop tumor growth. Therapeutic protons also generate short-lived activated nuclei of carbon, oxygen, and nitrogen atoms in patients as a result of atomic transmutations that are imaged by positron emission tomography (PET). We hypothesized that the transition of 18 O to 18 F in an 18 O-substituted nucleoside irradiated with therapeutic protons may result in the potential for combined diagnosis and treatment for cancer with proton therapy. Materials and Methods Reported here is a feasibility study with a therapeutic proton beam used to irradiate H 2 18 O to a dose of 10 Gy produced by an 85 MeV pristine Bragg peak. PET imaging initiated >45 minutes later showed an 18 F decay signal with T 1/2 of ∼111 minutes. Results The 18 O to 18 F transmutation effect on cell survival was tested by exposing SQ20B squamous carcinoma cells to physiologic 18 O-thymidine concentrations of 5 μM for 48 hours followed by 1- to 9-Gy graded doses of proton radiation given 24 hours later. Survival analyses show radiation sensitization with a dose modification factor (DMF) of 1.2. Conclusions These data support the idea of therapeutic transmutation in vitro as a biochemical consequence of proton activation of 18 O to 18 F in substituted thymidine enabling proton radiation enhancement in a cancer cell. 18 O-substituted molecules that incorporate into cancer targets may hold promise for improving the therapeutic window of protons and can be evaluated further for postproton therapy PET imaging.