Abstract Neurotensin (NTS) is a 13 amino acid peptide which is present in many lung cancer cell lines (Moody et al., Life Sci 1985; 36: 1727). High expression of the NTS receptor 1 (R1) is associated with poor survival of non-small cell lung cancer (NSCLC) patients (Alfano et al., Clin Cancer Res 2010; 16: 4401). NTS stimulates but the NTSR1 antagonist SR48692 inhibits the proliferation of NSCLC cells (Moody et al., Life Sci 2014; 100: 25). NTSR1 regulates NSCLC proliferation by transactivation of the EGFR. The cellular signaling mechanisms of the NTSR1 were investigated using NSCLC cells. Using NCI-H838 or A549 cells, addition of NTS or NTS(8-13) but not NTS(1-8) increased tyrosine phosphorylation of P-EGFR, P-HER2 and P-ERK 4-, 3- and 2-fold, respectively. The increase in EGFR and HER2 transactivation caused by NTS addition to NSCLC cells was blocked by SR48692, gefitinib (EGFR tyrosine kinase inhibitor (TKI)), lapatinib (EGFR and HER2 TKI), N-acetyl cysteine ((NAC) is an antioxidant), tiron (reactive oxygen species (ROS) scavenger) or diphenyleneiodonium (DPI is an inhibitor of NOX and DUOX enzymes). NTS increased ROS in NSCLC cells which was inhibited by NAC, tiron or DPI. NTS or NTS(8-13) stimulate NSCLC colony growth but SR48692, gefitinib, lapatinib or DPI inhibit growth. The results indicate that the NTSR1 regulates in a ROS-dependent manner the formation of EGFR homodimers or EGFR-HER2 heterodimers in NSCLC cells. Citation Format: Terry W. Moody, Lingaku Lee, Robert T. Jensen. Reactive oxygen species are essential for neurotensin receptors to regulate EGFR and HER2 transactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1007.