CD66a (human homolog of rat cell-cell adhesion molecule, also known as biliary glycoprotein) is a cell surface protein of the immunoglobulin family. CD66a has been shown to mediate homotypic cell adhesion. Aside from this, no other functions of this molecule have been demonstrated. We have observed previously that CD66a protein expression is lost in most prostate tumors, suggesting that the down-regulation of CD66a is associated with the abnormal growth of prostate cells. CD66a is homologous (65% identity) to rat cell-cell adhesion molecule, which has been shown to have tumor-suppressive activity. This homology suggests the possibility that CD66a might also be a tumor suppressor. In this report, we show that restoring CD66a expression in DU145 human prostate cancer cells by adenovirus (Ad)-mediated gene transfer dramatically altered the malignant phenotype of these cells, as evidenced by their reduced ability to form tumors in a xenograft animal model. This result suggests that loss of CD66a protein plays an important role in the development of prostate cancer, and that restoring CD66a expression might provide an effective treatment for prostate cancer. We further explored the possibility of using Ad vectors to deliver CD66a as a potential therapeutic agent for prostate cancer. Direct injection of Ad-CD66a, an Ad vector carrying the CD66a gene, into DU145 tumors in mice significantly suppressed the growth of these tumors. This antitumor activity of CD66a was found to be dose-dependent. These results suggest that CD66a has tumor-suppressive activity and that Ad-CD66a is a potential therapeutic agent for prostate cancer treatment.