Previous studies in experimental models have demonstrated that the transduction of human or murine melanoma cells with the co-stimulatory B7-1 molecule induces effective antitumor immune responses. In order to develop B7-1 gene transfer as a therapeutic tool in the clinical management of melanoma, efficient means of in vivo gene transfer must be used. To this end we evaluated in vitro and in vivo immune responses associated with adenoviral transduction of murine and human melanoma cells with B7-1. Adenovirus-mediated transduction of human and murine melanoma cells with B7-1 leads to high-level transgene expression in vitro and in vivo and does not affect MHC class I and II expression. Adenovirus-delivered B7-1 induced antitumor immune responses, on the basis of observations that human melanoma cells transduced to express human B7-1 were able to co-stimulate allogeneic and autologous T cells to proliferate and that murine melanoma K1735 cells transduced to express murine B7-1 were rejected by syngeneic, immunocompetent mice. By contrast, intratumoral injection of an adenovirus encoding murine B7-1 failed to eliminate established murine melanoma (K1735) despite high-level transgene expression in tumor cells. Potent T cell inhibitory factor(s) secreted by both K1735 cells and select human melanoma cells may contribute to the failure to achieve protection in this setting. Thus, immune inhibitory melanoma-derived factors need to be taken into account when considering the clinical use of B7-1 immunotherapy.