Fructose-1,6-diphosphate (FDP), a physiologic product of glycolysis, has been a useful therapeutic agent in mitigating the harmful effects of endotoxemia and ischemia/reperfusion. Since nitric oxide (NO) production has been implicated in tissue damage caused by endotoxemia and ischemia/reperfusion, we showed that 1, 5, or 10 mM FDP reduced the generation of NO by 17, 42, and 68%, respectively, in lipopolysaccharide (LPS)-treated macrophages. During these studies, we observed that LPS exposure resulted in macrophage aggregation, a cytologic finding that was markedly reduced in macrophages simultaneously treated with FDP. A recent study showed that sonicates of Mycobacterium avium aggregate macrophages and initiate apoptosis. Because apoptosis of NO-producing macrophages is related to diminished mitochondrial respiration, we hypothesized that FDP would maintain cellular reducing equivalents (pyridine nucleotides) in LPS-exposed macrophages. Rat alveolar macrophages (50,000 cells/well) were exposed to 10 ng of LPS in the presence or absence of 1, 5, or 10 mM FDP. After 24 hours, the ability of cells to reduce MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl bromid) was measured spectrophotometrically. The ability to reduce MTT was maintained in non-treated macrophages cultured between 0 and 24 hours. The mean absorbances (A550) of treated cells as a percentage of non-treated cells were: LPS = 28 ± 6%; 1 mM FDP and LPS = 73 ± 5%*; 5 mM FDP and LPS = 60 ± 7%*; and 10 mM FDP and LPS = 56 ± 8% (* =P<0.05 v. LPS, mean ± SEM, n = 5). Incubations with 5 mM fructose or 5 mM sodium phosphate, possible products of FDP catabolism, had A550 percentages that were 41 ± 7% and 28 ± 5%, respectively, of the non-treated cells. Treatment of macrophages with LPS and N-monomethyl-L-arginine, a competitive inhibitor of LPS-inducible nitric oxide synthase, resulted in A550 percentages that were similar to macrophages treated with LPS alone. Taken together, we conclude that FDP maintains cellular reducing equivalents in rat alveolar macrophages exposed to endotoxin, and this action appears to be separate from its ability to inhibit the production of NO. Since it is thought that phosphorylated sugars do not cross cell membranes, we propose the FDP effect is localized to the calcium pump fraction of plasma membrane where glycolytic enzymes produce NADH and ATP in the presence of FDP and cofactors. In summary, intravenous FDP may be useful in the clinical setting as a cytoprotective agent when the integrity of macrophages is compromised by endotoxin exposure.