3'-Deoxy-3'-[(18)F]fluorothymidine positron emission tomography ([(18)F]FLT PET) and diffusion weighted magnetic resonance imaging (DW-MRI) are promising approaches to monitor tumor therapy response. Here, we employed these two imaging modalities to evaluate the response of lung carcinoma xenografts in mice after gemcitabine therapy. Caliper measurements revealed that H1975 xenografts responded to gemcitabine treatment, whereas A549 growth was not affected. In both tumor models uptake of [(18)F]FLT was significantly reduced 6 h after drug administration. Based on the gemcitabine concentration and [(18)F]FLT excretion measured, this was presumably related to a direct competition of gemcitabine with the radiotracer for cellular uptake. On d1 after therapy [(18)F]FLT uptake was increased in both models, which was correlated with thymidine kinase 1 (TK1) expression. 2 d and 3 d after drug administration [(18)F]FLT uptake as well as TK1 and Ki67 expression were unchanged. A reduction in [(18)F]FLT in the responsive H1975 xenografts could only be noted on d5 of therapy. Changes in ADCmean in A549 xenografts 1 d or 2 d after gemcitabine did not seem to be of therapy-related biological relevance since they were not related to cell death (assessed by caspase-3 immunohistochemistry and cellular density) or tumor therapy response. Taken together, in these models, early changes of [(18)F]FLT uptake in tumors reflected mechanisms such as competing gemcitabine uptake or gemcitabine-induced TS inhibition and only reflected growth inhibitory effects at a later time point. Hence, the time point for [(18)F]FLT PET imaging of tumor response to gemcitabine is of crucial importance.