Lipopolysaccharide (LPS) induces the metal-binding proteins, metallothionein and ceruloplasmin, in the lung. This is a potential salvage mechanism for zinc and copper when they are purged from proteins by the oxidants generated during endotoxemia. Since the expression of metal-binding proteins is rapid (< 2 hours), we postulated that unbound Zn and Cu might inhibit the expression of alveolar macrophage (Mφ) genes induced by LPS. A candidate alveolar Mφ gene maximally induced by LPS within 8 hours of exposure is nitric oxide (NO) synthase. We tested this hypothesis by stimulating a rat alveolar Mφ cell line (NR8383) with LPS (50 ng) and interferon-γ (IFN-γ = 100 U). Thereafter, we measured NO production as nitrite [Griess reaction] at 24 hours. Zinc chloride (10 - 200μM) or copper chloride (10 - 100 μM) were added either with the stimuli(0 hour) or 8 hours after LPS and IFN-γ treatment. Unstimulated Mφ produced <0.5 μM nitrite, while stimulated Mφ generated 57±4μM nitrite. Stimulated Mφ exposed to 200 μM Zn at 0 hour produced 1.9±0.4 μM nitrite (P<0.05, n = 4), while 10 μM Zn added at 0 hour did not effect nitrite generation. Stimulated Mφ exposed to 100 μM Cu at 0 hour produced 9.6±0.3 μM nitrite(P<0.05), while 10 μM Cu added at 0 hour enhanced nitrite generation (82±3 μM, P<0.05). Intermediate concentrations of Zn or Cu added at 0 hour had less inhibitory effects. Inhibitory concentrations of Zn or Cu added at 8 hours resulted in rates of nitrite production akin to those for Mφ treated with LPS and IFN-γ without metal exposure. Northern blot analyses of mRNA expression for inducible NO synthase using stimulated Mφ treated with 100 or 200 μM Zn at 0 or 8 hours showed an inhibition of induction in 0 hour cells, but none in 8 hour specimens. RNA was isolated 24 hours after stimulation. We conclude that free Zn and Cu can inhibit induction of NO synthase in rat alveolar macrophages. We speculate that during endotoxemia, Zn and Cu are sequestered by metallothionein and ceruloplasmin, thereby favoring NO production.