Our preclinical work showed a dramatic synergy between interleukin-12 (IL-12) and trastuzumab for stimulation of natural killer cell cytokine secretion. We aimed to determine the safety profile of IL-12 when given in combination with trastuzumab and paclitaxel to patients with metastatic HER2-overexpressing cancers. Paclitaxel was given i.v. at 175 mg/m² every 3 weeks. Trastuzumab was given on day 1 each week (4 mg/kg initially and 2 mg/kg thereafter) in combination with injections of IL-12 on days 2 and 5 starting in cycle 2. This trial accrued 21 patients with metastatic HER2-positive tumors (breast, 7; colon, 6; esophagus, 4; stomach, 2; pancreas, 1; thyroid, 1). The IL-12 component was dose-escalated in cohorts of three patients. The dose-limiting toxicity was grade 3 fatigue at the 300 ng/kg dose level in two patients. The recommended phase II dose was 200 ng/kg administered s.c. There was one complete response in a patient with breast cancer, partial responses in 4 patients (breast, 2; esophageal, 2), and stabilization of disease lasting 3 months or greater (SD) in 6 other patients. All but one response occurred in patients with HER2 3+ disease. Two SD patients completed 1 year of therapy. Ten patients had progressive disease. There was increased activation of extracellular signal–regulated kinase in peripheral blood mononuclear cells and increased levels of IFN-γ and several chemokines in patients with clinical benefit (complete response, partial response, or SD), but not in patients with progressive disease. IL-12 in combination with trastuzumab and paclitaxel therefore exhibits an acceptable toxicity profile and has activity in patients with HER2-overexpressing cancers. [Mol Cancer Ther 2009;8(11):2983–91]