Background: Smoothened (SMO) gene somatic mutations activate hedgehog signaling in basal cell cancers (BCC) and medulloblastomas but have not been reported in NSCLC. We detected somatic and germline SMOmutations in NSCLC patients (A-C) and sought to characterize the mutations further. Methods: We performed tumor/blood germline sequencing of SMO mutations, familial germline mutation testing (saliva specimens), somatic mutation analysis of TCGA lung carcinoma datasets, and germline mutation analysis of the TCGA and 3 additional large cohorts (n = 1933). To evaluate the functional significance of SMOP641A, HCC4011 lung cancer cells were transfected with a wild-type SMO or SMOP641A expression vector and a predictive model was created. Results: NSCLC SMOP641A in vitro activated the hedgehog pathway, and vismodegib/cyclopamine inhibited tumor cell growth. Structural modeling suggests that SMO P641A induces conformational changes and disrupts PTCH-SMO interaction leading to constitutive activation. In the NSCLC TCGA databases, somatic SMO mutations occur 1.7%. In the overall TCGA database, germline SMO P641A occurred in 0.11% of cancer patients (multiple cancers) compared with 0% in cancer-free individuals. Patient A (never-smoking SCC) had a 46% RECIST reduction within 6 weeks for 6 months on vismodegib. His 3-generation family pedigree identified germline SMOP641A in one daughter (who developed BCC early). Two additional NSCLC patients (B – germline P641A and C –M525L received vismodegib; B initially stabilized but stopped vismodegib after 14 weeks for toxicity while patient C had no response. Conclusions:SMO mutations are targetable, potentially heritable, oncogenic drivers in NSCLC and other cancers. Tumor genetic profiling should consider including SMO gene, especially in never-smoking lung SCC patients. Additional studies are needed to define the role of germline/somatic SMO alterations in promoting carcinogenesis, interactions with P53 alterations, and the responsiveness of different SMO mutations to hedgehog inhibitors. Currently, the ongoing ECOG-ACRIN MATCH study (NCT02465060) treats SMO/PTCH mutated patients with vismodegib.