Gram-negative pneumonia and bacteremia are life-threatening events seen in intensive care units. Earlier studies have indicated that fructose-1,6-diphosphate (FDP) enhances the survival of animals given intravenous lipopolysaccharide (LPS), but its mechanism of action is unknown. Because the production of nitric oxide (NO) is associated with septic shock, we hypothesized that FDP would inhibit the generation of NO by macrophages. Cultured rat alveolar macrophages (500,000 cells per well) were treated with 1, 5, or 10 mM FDP for 4 hours before adding 100 ng/ml of LPS. After 24 hours, the NO metabolite, nitrite, was measured in culture supernatants using the Griess reaction. The micromolar concentrations of nitrite were: Non-treated = 2.3 ± 0.6; LPS = 15.9 ± 1.2*; 1 mM FDP + LPS = 13.2 ± 0.6; 5 mM FDP + LPS = 9.2 ± 0.8**; and 10 mM FDP + LPS = 5.1 ± 1.0** (* = P<0.01 v. Non-treated; ** = P<0.01 v. LPS alone; mean ± S.E.M., n = 6). Consistent with differences in the production of nitrite at 24 hours, Northern blot analyses showed a proportional reduction in mRNA for inducible NO synthase (iNOS) when macrophages were treated with 1, 5, or 10 mM FDP + LPS compared to LPS alone. Neither fructose nor phosphate, possible catabolic products of FDP, affected the production of NO or iNOS expression. Mobility shift assays indicated that FDP did not inhibit LPS-mediated activation of nuclear factor kappa B. Studies of macrophage viability performed using trypan blue dye exclusion showed that the effects of FDP were not related to cellular toxicity. An ion-selective electrode was used to measure calcium in culture supernatants. Five and 10 mM FDP and 5 mM sodium phosphate significantly reduced ionized calcium in culture supernatants. However, sodium phosphate did not affect LPS-mediated iNOS expression or NO production, and therefore, the inhibitory effect of FDP appears not to be associated with reductions in extracellular calcium. In conclusion, fructose-1,6-diphosphate, a glycolytic intermediate with potential clinical use, may alleviate the adverse effects of LPS via an inhibition of nitric oxide production.