The biochemical integrity of hepatocellular mitochondria was investigated in rats treated with small doses of human recombinant tumor necrosis factor-alpha (Hur-TNF;50-100 micrograms/kg/d injected intraperitoneally for 5 d) by measuring the activities of three mitochondrial enzymes, glutamate dehydrogenase, succinate dehydrogenase and malate dehydrogenase. The activity of glutamate dehydrogenase (a mitochondrial matrix enzyme) was 20% to 34% lower than that of control rats (P = 0.02 to 0.0003). The activities of succinate dehydrogenase (an inner mitochondrial membrane enzyme) and malate dehydrogenase (a mitochondrial matrix and cytosolic enzyme) showed no significant difference. The effect of TNF on serum amino acid composition was studied using pair-fed, weight-matched partners to eliminate any effect of the reduction of food intake due to TNF treatment. The results for the TNF-treated rats showed a significant (P < 0.05) increase in the concentration of 12 of the 21 amino acids measured (range = 33% to 140%). Of these, major increases were observed in the urea cycle intermediates, ornithine (140%) and arginine (59%), as well as proline (94%), alanine (41%), valine (61%), leucine (64%), isoleucine (63%), and aspargine (71%). Since previous studies have shown that the treatment of rats with the same low doses of TNF did not cause any change in mitochondrial ultrastructure detectable by electron microscopy, these results suggest that significant biochemical changes in amino acid metabolism occur as a result of a decrease in mitochondrial glutamate dehydrogenase activity.