TNF antagonists may offer therapeutic potential in solid tumors, but patients who have high serum levels of TNF-alpha fail to respond to infliximab, suggesting consumption of the circulating antibody and loss of transmembrane TNF-alpha (tmTNF-alpha) on tumors by ectodomain shedding. Addressing this possibility, we developed a monoclonal antibody (mAb) that binds both full-length tmTNF-alpha and its N-terminal truncated fragment on the membrane after tmTNF-alpha processing but does not cross-react with soluble TNF-alpha. We documented high levels of tmTNF-alpha expression in primary breast cancers, lower levels in atypical hyperplasia or hyperplasia, but undetectable levels in normal breast tissue, consistent with the notion that tmTNF-alpha is a potential therapeutic target. Evaluations in vitro and in vivo further supported this assertion. tmTNF-alpha mAb triggered antibody-dependent cell-mediated cytotoxicity against tmTNF-alpha-expressing cells but not to tmTNF-alpha-negative cells. In tumor-bearing mice, tmTNF-alpha mAb delayed tumor growth, eliciting complete tumor regressions in some mice. Moreover, tmTNF-amAb inhibited metastasis and expression of CD44v6, a prometastatic molecule. However, the antibody did not activate tmTNF-alpha-mediated reverse signaling, which facilitates tumor survival and resistance to apoptosis, but instead inhibited NF-kappa B activation and Bcl-2 expression by decreasing tmTNF-alpha-positive cells. Overall, our results established that tmTNF-alpha mAb exerts effective antitumor activities and offers a promising candidate to treat tmTNF-alpha-positive tumors, particularly in patients that are nonresponders to TNF antagonists.