It is well documented that nitric oxide (NO) is an effector molecule of macrophage-mediated tumor cell toxicity in vitro; however, little is known about the role of NO in the antitumor immune response in vivo. We have developed a treatment protocol using lipid A. We have investigated the effects of lipid A on inducible NO synthase (NOS II) expression and evolution inside tumors during the course of treatment. Lipid A (OM-174) treatment induced tumor regression in rats bearing established colon tumors. Furthermore, NO was synthesized and secreted inside the tumors of lipid A-treated rats, as demonstrated by the increase of NOS II mRNA and NOS II content in the tumors, as well as of NOS II activity and NO production. During treatment, NOS II was localized in tumor cells only. Lipid A had no direct effect on tumor cells in vitro, while the combination of interferon gamma (IFN-gamma) plus interleukin-1 beta (IL-1beta) induced production of NO by tumor cells which was cytostatic. The content of IFN-gamma and IL-1beta in tumors was enhanced during lipid A treatment; this is in agreement with an indirect effect of lipid A in vivo via the IFN-gamma and IL-1beta pathways.