3'-Deoxy-3'-[$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]fluorothymidine positron emission tomography ([$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]FLT-PET) and diffusion-weighted MRI (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 [$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]FLT was significantly reduced 6 hours after drug administration. On the basis of the gemcitabine concentration and [$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]FLT excretion measured, this was presumably related to a direct competition of gemcitabine with the radiotracer for cellular uptake. On day 1 after therapy, [$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]FLT uptake was increased in both models, which was correlated with thymidine kinase 1 (TK1) expression. Two and 3 days after drug administration, [$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]FLT uptake as well as TK1 and Ki67 expression were unchanged. A reduction in [$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]FLT in the responsive H1975 xenografts could only be noted on day 5 of therapy. Changes in ADC in A549 xenografts 1 or 2 days after gemcitabine did not seem to be of therapy-related biological relevance as they were not related to cell death (assessed by caspase-3 IHC and cellular density) or tumor therapy response. Taken together, in these models, early changes of [$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]FLT uptake in tumors reflected mechanisms, such as competing gemcitabine uptake or gemcitabine-induced thymidylate synthase inhibition, and only reflected growth-inhibitory effects at a later time point. Hence, the time point for [$\mathrm{<mrow\; data-mjx-texclass="ORD">18</mrow>}$F]FLT-PET imaging of tumor response to gemcitabine is of crucial importance.