We evaluated the ability of the PET imaging agent ⁸⁹Zr-trastuzumab to delineate HER2-positive gastric cancer and to monitor the pharmacodynamic effects of the epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor afatinib. Using ⁸⁹Zr-trastuzumab, ¹⁸F-FDG, or 3′-deoxy-3′-¹⁸F-fluorothymidine (¹⁸F-FLT PET), we imaged HER2-positive NCI-N87 and HER2-negative MKN74 gastric cancer xenografts in mice. Next, we examined the pharmacodynamic effects of afatinib in NCI-N87 xenografts using ⁸⁹Zr-trastuzumab and ¹⁸F-FDG PET and comparing imaging results to changes in tumor size and in protein expression as monitored by Western blot and histologic studies. Although ¹⁸F-FDG uptake in NCI-N87 tumors did not change, a decrease in ⁸⁹Zr-trastuzumab uptake was observed in the afatinib-treated versus control groups (3.0 ± 0.0 percentage injected dose per gram (%ID/g) vs. 21.0 ± 3.4 %ID/g, respectively; P < 0.05). ⁸⁹Zr-trastuzumab PET results corresponded with tumor reduction, apoptosis, and downregulation of HER2 observed on treatment with afatinib. Downregulation of total HER2, phosphorylated (p)-HER2, and p-EGFR occurred within 24 h of the first dose of afatinib, with a sustained effect over 21 d of treatment. Afatinib demonstrated antitumor activity in HER2-positive gastric cancer in vivo. ⁸⁹Zr-trastuzumab PET specifically delineated HER2-positive gastric cancer and can be used to measure the pharmacodynamic effects of afatinib.