Objectives To investigate the changes in tumour proliferation (using FLT), metabolism (using FDG), and hypoxia (using F-miso) during curative (chemo-) radiotherapy (RT) in patients with non-small-cell lung cancer (NSCLC). Patients and methods Thirty PET scans were performed in five patients (4 males, 1 female) that had histological proof of NSCLC and were candidates for curative-intent RT. Three PET-CT (Biograph S16, Siemens) scans were performed before ( t 0) and during (around dose 46 Gy, t 46) RT with minimal intervals of 48 h between each PET-CT scan. The tracers used were 18fluoro-2deoxyglucose (FDG) for metabolism, 18fluorothymidine (FLT) for proliferation, and 18F-misonidasole (F-miso) for hypoxia. The 3 image sets obtained at each time point were co-registered (rigid: n = 9, elastic: n = 1, Leonardo, TrueD, Siemens) using FDG PET-CT as reference. VOIs were delineated (40% SUV max values were used as a threshold) for tumours and lymph nodes on FDG PET-CT, and they were automatically pasted on FLT and F-miso PET-CT images. ANOVA and correlation analyses were used for comparison of SUV max values. Results Four tumours and twelve nodes were identified on initial FDG PET-CT images. FLT SUV max values were significantly lower ( p < 0.0006) at t 46 in both tumours and nodes. The decrease in FDG SUV max values had a trend towards significance ( p = 0.048). F-Miso SUV max values were significantly higher in tumours than in nodes ( p = 0.02) and did not change during radiotherapy ( p = 0.39). A significant correlation was observed between FLT and FDG uptake ( r = 0.56, p < 10 −4) when all data were pooled together, and they remained similar when the before and during RT data were analysed separately. FDG and F-miso uptakes were significantly correlated ( r = 0.59, p = 0.0004) when all data were analysed together. The best fit was obtained after adjusting for lesion type (tumour vs. node). This correlation was observed for the SUV max measured during RT ( r = 0.70, p = 0.008) but not for the pre-RT data ( r = 0.19, p = 0.35). The weak correlation between FLT and F-miso uptakes only became significant ( r = 0.66, p = 0.002) when the analysis was restricted to the data acquired during RT. Conclusion Three different PET acquisitions can be performed quasi-simultaneously (4–7 days) before and during radiotherapy in patients with NSCLC. Our results at 46 Gy suggest that a fast decrease in the proliferation of both tumours and nodes exists during radiotherapy with differences in metabolism (borderline significant decrease) and hypoxia (stable).