Head and neck squamous cell carcinomas (HNSCC) represent a group of metastasizing tumors with a high mortality rate in man and animals. Since the biomolecule ozone was found to inhibit growth of various carcinoma cells in vitro we here applied the highly aggressive and lethal VX2 carcinoma HNSCC tumor model of the New Zealand White rabbit to test whether ozone exerts antitumorous effects in vivo. Therapeutic insufflation of medical ozone/oxygen (O3/O2) gas mixture into the peritoneum (O3/O2-pneumoperitoneum) at an advanced stage of tumor disease led to a survival rate of 7/14 rabbits. Six of the seven surviving rabbits presented full tumor regression and the absence of local or distant lung metastases. Insufflation of pure oxygen (O2) resulted in a survival rate of 3/13 animals accompanied by full tumor remission in 2 of the 3 surviving animals. Of the 14 sham-treated animals only 1 had spontaneous tumor remission and survived. No adverse effects or changes in standard blood parameters were observed after repeated intraperitoneal insufflations of the O3/O2 or O2 gas. Animals with O3/O2-induced tumor eradication developed tolerance against reimplantation of the VX2 tumor. This could be reversed by immune suppression with a combination of dexamethasone and cyclosporin A suggesting an antitumorous effect of O3/O2-mediated activation of the body's own immunosurveillance. Although the exact mechanisms of action are still unclear the present data point to O3/O2-pneumoperitoneum as a promising new strategy in anticancer therapy. © 2008 Wiley-Liss, Inc.