Cryosurgery is a minimally invasive cancer treatment using cryogenic temperatures. Intraoperative monitoring of iceball growth is an advantage of the treatment. However, whereas the iceball can be easily visualized, destruction within the iceball is incomplete and the means to monitor the "kill zone" are urgently needed. Recently, we have shown the ability of tumor necrosis factor-α (TNF-α) to enhance destruction within an iceball. To avoid systemic toxicity, we delivered TNF-α selectively to the tumor by a gold nanoparticle of 30-nm diameter (CYT-6091) tagged with TNF-α and thiol-derivatized polyethylene glycol. Using a dorsal skin fold chamber (DSFC) in a nude mouse, both normal skin and human prostate carcinoma (LNCaPP ro 5) were pretreated with soluble TNF-α (topically or i.v.) or CYT-6091 (i.v.) and frozen after 4 h. The cryolesion was assessed after 3 days by comparing histologic necrosis with perfusion defects. Hind limb tumors were also treated by visibly encompassing the tumor with an iceball and assessing gross changes over time. A 5-μg dose of soluble TNF-α or CYT-6091 increased the temperature threshold of necrosis in the tumor in the DSFC from -14.0 ± 1.6°C (n = 6) to 0.9 ± 1.5°C (n = 6) and -1.5 ± 3.7°C (n = 6), respectively. In hind limb tumors, the same dose resulted in significant tumor shrinkage and remission in 2 of 8 (for soluble TNF-α) and in 3 of 8 (for CYT-6091). The nanoparticle alone group without TNF-α increased the temperature threshold of necrosis to -7.0 ± 2.3°C in the tumor in the DSFC and more shrinkage of the tumor in the hind limb when compared with cryo alone treatment. Systemic toxicity was noted in all soluble TNF-α groups but none with CYT-6091. These results suggest that it is possible to destroy all of a tumor within an iceball by preincubation with TNF-α and systemic toxicity can be avoided by CYT-6091.