Purpose Positron emission tomography (PET) using 18 F-3′-deoxy-3′-fluorothymidine ([ 18 F]FLT) allows noninvasive monitoring of tumour proliferation. For serial imaging in individual patients, good reproducibility is essential. The purpose of the present study was to evaluate the reproducibility of quantitative [ 18 F]FLT measurements. Methods Nine patients with non-small-cell lung cancer (NSCLC) and six with head-and-neck cancer (HNC) underwent [ 18 F]FLT PET twice within 7 days prior to therapy. The maximum pixel value (SUV max ) and a threshold defined volume (SUV 41% ) were defined for all delineated lesions. The plasma to tumour transfer constant (K i ) was estimated using both Patlak graphical analysis and nonlinear regression (NLR). NLR was also used to estimate k 3 , which, at least in theory, selectively reflects thymidine kinase 1 activity. The level of agreement between test and retest values was assessed using the intraclass correlation coefficient (ICC) and Bland-Altman analysis. Results All primary tumours and >90% of clinically suspected locoregional metastases could be delineated. In total, 24 lesions were defined. NLR-derived K i , Patlakderived K i , SUV 41% and SUV max showed excellent reproducibility with ICCs of 0.92, 0.95, 0.98 and 0.93, and SDs of 16%, 12%, 7% and 11%, respectively. Reproducibility was poor for k 3 with an ICC of 0.43 and SD of 38%. Conclusion Quantitative [ 18 F]FLT measurements are reproducible in both NSCLC and HNC patients. When monitoring response in individual patients, changes of more than 15% in SUV 41% , 20-25% in SUV max and Patlak-derived K i , and 32% in NLR3k-derived K i are likely to represent treatment effects.