The effect of human recombinant tumor necrosis factor (TNF)-alpha on enzymes of gluconeogenesis in the rat was investigated by determining the activity of glucose 6-phosphatase, fructose 1,6-diphosphatase (FDP), and phosphoenolpyruvate carboxykinase in the liver and kidney of fed and fasted rats. The activity of transaldolase in the pentose phosphate pathway was also measured. Starvation of rats for 24 hr resulted in a 1.6- to 3.1-fold increase in liver and kidney glucose 6-phosphatase and phosphoenolpyruvate carboxykinase (P less than or equal to 0.05), a decrease in liver and kidney FDP (P less than 0.002), and an increase in liver and kidney transaldolase (P = 0.0001). Injection of 50 and 100 micrograms/kg/day of TNF for 5 days resulted in a significant (P less than or equal to 0.03) decrease in kidney FDP only. Injection of 100 micrograms/kg/day of TNF for 5 days with a 24-hr fast on Day 5 resulted in a significant (P = 0.04) increase in liver transaldolase, and a significant decrease in kidney FDP and phosphoenolpyruvate carboxykinase. Comparison of the enzyme activities of rats injected with 100 micrograms/kg/day of TNF for 5 days with those of their pair-fed control partners revealed additionally a significant decrease in glucose 6-phosphatase in the liver (P less than 0.001). It is concluded that TNF administration in the rat has different effects on the enzymes of gluconeogenesis in the liver and kidney, and these effects differ from those seen in starved or tumor-bearing rats.