Generation of antitumor immunity by adenoviral gene transfer of interleukin-12 (IL-12) is a very promising concept in cancer gene therapy. Systemically, IL-12 has provoked toxic side effects at therapeutically relevant doses. Native IL-12 lacks effectiveness in clinical trials even when expressed intratumorally from adenoviral vectors. Our strategy was to increase the therapeutic efficacy of IL-12 by expressing a fusion protein of its two subunits (scIL-12) in an adenoviral vector and to evaluate the effects after intratumoral administration. In a rat model of hepatocellular carcinoma, this vector revealed antitumor effects even at a low dosage of 4.6 x 10(5) i.u. in a dose-dependent manner. Long-term antitumor effects were determined at 2.3 x 10(6) and 2.3 x 10(7) i.u. per animal, resulting in 82% and 90% surviving animals, respectively. Magnetic resonance imaging (MRI) enabled individual tumor size follow-up and revealed the scIL-12 effects on large tumors. Treating one hepatic lesion also led to tumor elimination in a second non-treated hepatic lesion. Animals rechallenged with tumor cells remained tumor-free. Compared to studies applying native IL-12, our data show that the fusion of IL-12 subunits provides approximately 1000-fold higher biological activity. As a consequence of the observed gain in activity, scIL12 promises a substantially improved antitumor efficacy and safety profile of intratumoral adenoviral IL-12 immunotherapy, supporting its clinical use.