Screening for oncogenes has mostly been performed by in vitro transformation assays. However, some oncogenes might not exhibit their transforming activities in vitro unless putative essential factors from in vivo microenvironments are adequately supplied. Here, we have developed an in vivo screening system that evaluates the tumorigenicity of target genes. This system uses a retroviral high-efficiency gene transfer technique, a large collection of human cDNA clones corresponding to ˜ 70% of human genes and a luciferase-expressing immortalized mouse mammary epithelial cell line (NMuMG-luc). From 845 genes that were highly expressed in human breast cancer cell lines, we focused on 205 genes encoding membrane proteins and/or kinases as that had the greater possibility of being oncogenes or drug targets. The 205 genes were divided into five subgroups, each containing 34–43 genes, and then introduced them into NMuMG-luc cells. These cells were subcutaneously injected into nude mice and monitored for tumor development by in vivo imaging. Tumors were observed in three subgroups. Using DNA microarray analyses and individual tumorigenic assays, we found that three genes, ADORA2B, PRKACB and LPAR3, were tumorigenic. ADORA2B and LPAR3 encode G-protein-coupled receptors and PRKACB encodes a protein kinase A catalytic subunit. Cells overexpressing ADORA2B, LPAR3 or PRKACB did not show transforming phenotypes in vitro, suggesting that transformation by these genes requires in vivo microenvironments. In addition, several clinical data sets, including one for breast cancer, showed that the expression of these genes correlated with lower overall survival rate.