The effect of treatment of rats with bacterial endotoxin on gluconeogenesis and the flux through pyruvate kinase, phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase and pyruvate dehydrogenase (PDH) was measured in isolated hepatocytes, prepared from animals starved for 18 h, incubated in the presence of 1 mM pyruvate. The lipopolysaccharide reduced gluconeogenesis by 50% and lowered the flux through pyruvate kinase, PEPCK and pyruvate carboxylase by comparable amounts. There was no effect of endotoxaemia on PDH flux, indicating that the lowered rate of gluconeogenesis is not the result of a redistribution of pyruvate metabolism between oxidation and carboxylation. The results confirm that a stimulation of pyruvate kinase activity following treatment with lipopolysaccharide is not involved in the inhibition of gluconeogenesis, but that the effect resides at the level of phosphoenolpyruvate formation. The most favoured mechanism for the inhibition of glucose synthesis is via an inhibition of PEPCK and subsequent feedback inhibition of pyruvate carboxylase, although a secondary effect at the level of the mitochondria and pyruvate carboxylase cannot be excluded.