Purpose: The goal of this study is to identify novel genes frequently silenced by promoter hypermethylation in lung cancer. Experimental Designs: Bioinformatic analysis was done to identify candidate genes significantly down-regulated in lung cancer. The effects of DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine on the expression of the candidate genes were determined. Methylated CpG sites in the promoters of the candidate genes were identified using bisulfite DNA sequencing. Methylation-specific PCR was developed and used to analyze DNA methylation in cell lines and clinical specimen. Pathologic and functional analyses were done to study the role of one candidate gene, receptor activity-modifying protein 2 (RAMP2), in suppressing lung cancer cell growth. Results: Among 54 candidate genes down-regulated in lung cancer, 31 were found to contain CpG islands in their promoters. Six of these 31 genes could be reactivated by 5-aza-2′-deoxycytidine in at least four of six lung cancer cell lines analyzed. Promoter hypermethylation of RAMP2, epidermal growth factor - containing fibulin-like extracellular matrix protein 1, and deleted in U Twenty Twenty cells was detected in 36% to 77% of 22 lung cancer cell lines and in 38% to 50% of 32 primary lung tumors, whereas hypermethylathion of these genes was rarely found in the matched normal samples. The methylation frequencies of these genes in lung cancer were similar to those of commonly used methylation markers, such as RAS association domain family protein 1A, p16, and methylguanine-DNA methyltransferase. Immunohistochemistry showed that RAMP2 was down-regulated in a majority of lung tumors, and RAMP2 down-regulation was correlated with high tumor grade. Ectopic expression of RAMP2 inhibited lung cancer cell growth and caused apoptotic cell death. Knockdown of RAMP2 by RNA interference stimulated cell proliferation. Conclusions: Studying the newly identified genes may provide new insight into lung tumorigenesis. These genes might be useful as molecular markers of lung cancer.