Purpose Oncogenic gene fusions involving the 3’ region of ROS1 kinase have been identified in various human cancers. In this study, we sought to characterize ROS1 fusion genes in non-small cell lung cancer (NSCLC) and establish the fusion proteins as drug targets. Experimental Design A NSCLC tissue microarray (TMA) panel containing 447 samples was screened for ROS1 rearrangement by fluorescence in-situ hybridization (FISH). This assay was also used to screen NSCLC patients. In positive samples, the identity of the fusion partner was determined through inverse-PCR and RT-PCR. In addition, the clinical utility of ROS1 inhibition was assessed by treating a ROS1-positive patient with crizotinib. The HCC78 cell line, which expresses the SLC34A2-ROS1 fusion, was treated with kinase inhibitors that have activity against ROS1. The effects of ROS1 inhibition on proliferation, cell-cycle progression, and cell signaling pathways were analyzed by MTS assay, flow cytometry, and western blotting. Results In the TMA panel, 5/428 (1.2%) evaluable samples were found to be positive for ROS1 rearrangement. Additionally, 1/48 patients tested positive for rearrangement, and this patient demonstrated tumor shrinkage upon treatment with crizotinib. The patient and one TMA sample displayed expression of the recently identified SDC4-ROS1 fusion, while two TMA samples expressed the CD74-ROS1 fusion and two others expressed the SLC34A2-ROS1 fusion. In HCC78 cells, treatment with ROS1 inhibitors was anti-proliferative and down-regulated signaling pathways that are critical for growth and survival. Conclusions ROS1 inhibition may be an effective treatment strategy for the subset of NSCLC patients whose tumors express ROS1 fusion genes.