Ebp1, an ErbB3 binding protein and downstream effector of the ErbB signaling network was shown to be a potent tumor suppressor in breast and prostate adenocarcinomas. We hypothesized that the inhibitory properties of the ebp1 gene could also be beneficial if ectopically expressed in salivary adenoid carcinoma. Salivary adenoid carcinoma cell line (ACC-M) cells were stably transfected with the full-length ebp1 cDNA sequence or the empty expression vector pcDNA3.1. Stable gene transfer was verified by Western blot analysis and reverse transcription (RT)-PCR. A significant reduction in cell proliferation, anchorage-independent growth, and a change in the cell cycle profile was observed in ebp1 transfectants. Matrigel assays demonstrated that the adenoid cystic carcinoma cell invasiveness was significantly reduced. A strong decrease in the metastatic potential of human adenoid cystic carcinoma cells in an experimental metastatic model was also observed. Our results suggest that ectopic expression of Ebp1 mediates multiple antitumor activities against adenoid cystic carcinoma cells and that ebp1 gene therapy might be a viable method suppressing malignant salivary adenoid tumors. © 2007 Wiley-Liss, Inc.