Bacterial lipopolysaccharide (LPS) has been recognized as one of the most potent activating signals for mouse peritoneal macrophages. In macrophages primed by interferon-gamma (IFN-gamma) or trehalose dimycolate (TDM), LPS induces NO synthase and the events associated with a high nitric oxide output: antitumor and antiparasitic activities. In the present report, it is shown that drugs (calcium ionophores or thapsigargin) which elevate the concentration of cytosolic calcium, [Ca2+]i, induce NO synthase and antitumor activities in primed macrophages, mimicking LPS action. Calcium ionophores and thapsigargin trigger NO synthase activity in macrophages primed in vivo by TDM, in thioglycollate-elicited macrophages primed in vitro by IFN-gamma, and in IFN-gamma-treated EMT6 adenocarcinoma cells. However, activation of TDM-primed macrophages by LPS does not seem to involve calcium fluxes: (i) no change in [Ca2+]i was detectable in TDM-primed macrophages loaded with Fura-2 and exposed to LPS, and (ii) activation of TDM-primed macrophages by LPS can be obtained in the presence of 4 mM EGTA. NO synthase expression is thus controlled in primed macrophages by two different pathways; calcium ionophores can replace LPS but do not act through the same intracellular cascade.