Abstract Erlotinib (Tarceva) targets the epidermal growth factor receptor (EGFR), which is commonly overexpressed in human cancers, including lung cancer. We show that erlotinib can be labeled with [11C] by reacting the normethyl precursor with [11C]-methyl iodide. By using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, two lung cancer cell lines (A549 and NCI358) were shown to be less sensitive to erlotinib compared with the lung cancer cell line HCC827. This correlated with higher expression and activity of the EGFR in HCC827 cells as compared with the less sensitive cell lines. Micro–positron emission tomography (PET) and biodistribution of erlotinib was performed with [11C]-erlotinib in nude mice bearing xenografts of A549, NCI358, and HCC827 cells. Dynamic micro-PET showed that HCC827 tumors had the highest [11C]-erlotinib uptake and retained the activity significantly longer as compared with A549 and NCI358 tumors. Biodistribution of [11C]-erlotinib in the xenograft models of lung cancer showed the highest accumulation in the liver. In mice carrying the sensitive cancer cells, the accumulation of [11C]-erlotinib was higher in tumors than in the other organs. In contrast, the drug accumulated to a comparable extent in tumors from the less sensitive cancer cells and the other organs. Uptake of [11C]-erlotinib in the tumors was 1.6%, 0.7%, and 3.7% (percentage of injected dose/g), in A549, NCI358, and HCC827 cells, respectively. We show for the first time that [11C]-erlotinib identifies erlotinib-sensitive tumors. These results pave the road for studies examining the benefit of [11C]-erlotinib PET in patients with lung tumors or other tumors overexpressing EGFR. [Cancer Res 2009;69(3):873–8]