Objective/Hypothesis: Adenoviral interleukin‐2 (AdV‐IL‐2) gene therapy has previously not proven effective in treating established murine oral cancer. We hypothesize that the intratumoral level of IL‐2 expression is a major limiting factor in treatment outcome. Methods: A microscopic disease and established oral cancer murine model was used to test this hypothesis. IL‐2 gene transfer was performed with a recombinant adenovirus vector. Results: Tumor cells were transduced in vitro with AdV‐IL‐2 and subsequently implanted into the floor of the mouth in C3H/ HeJ mice. IL‐2 expression in vitro ranged from 990 to 1,050 pg/10⁶ tumor cells. This microscopic disease treatment resulted in either complete tumor regression or a dramatic decrease in tumor progression. Cytolytic T‐cell (CTL) assays demonstrated a predominance of CD8‐specific, T‐cell–mediated tumor killing. Reducing IL‐2 expression by half with a mixture of 1:1 transduced to nontransduced tumor cells eliminated the antitumor effect and decreased the CTL response. These findings support the presence of a critical “threshold” of IL‐2 expression. Adenovirus repurification and amplification allowed isolation of a twofoldhigher‐titer AdV‐IL‐2 vector. Treatment of established tumors with the higher‐titer AdV‐IL‐2 at a new maximal dose of 1.4 × 10⁹ plaque‐forming units (pfu) increased in vivo IL‐2 expression to 1,127 pg/10⁶ cells and generated a significant antitumor response. Complete regression of established tumors, however, could not be achieved, and we noted a decrease in IL‐2 expression well below the threshold at 1 week after treatment. Upon repeat maximal AdV‐IL‐2 injection in vivo, a greater antitumor effect and increased CTL response was seen, but also, 28% of the animals died of IL‐2 toxicity. Conclusion: Although limited by expression and toxicity as a single‐treatment strategy for established tumors, AdV‐IL‐2 gene therapy should be considered a potential component of combination therapy strategies.