The present study evaluates the ability of a new drug carrier: nanocapsules of poly(D,L-lactide) containing muramyldipeptide-L-alanyl-cholesterol (MTP-Chol NC) to induce activation of mouse macrophage cell lines. MTP-Chol NC stimulated nitric oxide (NO) expression and tumor necrosis factor-alpha (TNF-alpha) production, these are two important mediators of macrophage-mediated cytotoxicity. The encapsulated form was more effective than free muramyldipeptide, at low immunomodulator concentrations. The dose-response curves were completely different for NO and TNF-alpha, implying different regulatory mechanisms. In RAW 264.7 cells, the addition of anti-TNF-alpha antibodies during the activation period did not affect the level of nitrite induced by MTP-Chol Nc and lipopolysaccharide. Therefore, autocrine stimulation by TNF-alpha did not contribute to NO production. On the other hand, the presence of an NO synthase inhibitor led to an increase in TNF-alpha secretion. In J774.A1 cells, which were activated by MTP-Chol NC and interferon-gamma, TNF-alpha production seemed to act as a second messenger. Thus, under certain conditions, NO can play a role in modulating the cytotoxic activities of mouse macrophages.