Interleukin-12 (IL-12) has potent antitumor activity, but its clinical application is limited by severe systemic toxicity, which might be alleviated by the use of membrane-anchored IL-12. In the present study, a new membrane-bound IL-12 containing murine single-chain IL-12 and B7-1 transmembrane and cytoplasmic domains (scIL-12-B7TM) was constructed and its efficacy in cancer treatment examined and its protective antitumor mechanism investigated. Intratumoral injection of an adenoviral vector encoding scIL-12-B7TM not only resulted in complete regression of a majority of local tumors, but also significantly suppressed the growth of distant, untreated tumors. Immunohistochemical staining demonstrated that both CD8+ T cells and CD4+ T cells contributed to the antitumor activity of scIL-12-B7TM. Our data demonstrate that cancer immunotherapy using membrane- bound IL-12 has the advantage of minimizing systemic IL-12 levels without compromising its antitumor efficacy.