Because of differences in the pattern of enzyme activities involved in glycolysis and gluconeogenesis between rat and guinea pig liver, the regulation of gluconeogenesis in guinea pig liver was studied.The following results were obtained: 1. Starvation increases the capacity for gluconeogenesis from pyruvate in vivo. 2. Ghconeogenesis from L-lactate and from pyruvate in isolated perfused livers is greater in the guinea pig than in the rat, whereas gluconeogenesis from other precursors is similar in both species.3. Long chain and medium chain fatty acids inhibit gluconeogenesis from L-lactate in isolated perfused guinea pig liver but stimulate gluconeogenesis from L-lactate in isolated perfused rat liver. I n the guinea pig liver the difference does not result from a lower rate of fatty acid oxidation but from a greater reduction in the cytoplasmic NAD+/NADH system.During enhanced fatty acid oxidation in guinea pig liver and in rat liver, similar changes in the concentrations of both acetyl-S-CoA and CoA-XH are observed.With high concentrations of pyruvate as the gluconeogenic precursor, both fatty acids and ethanol lead to a slight stimulation of glucose formation by supplying hydrogen equivalents to the cytosolic NAD+/NADH system. 4. Both glucagon and dibutyryl-Ado-3' ; 5'-P stimulate ureogenesis and ketogenesis in isolated perfused guinea pig liver. However, the stimulation of gluconeogenesis by glucagon and Ado-3';5'-P normally observed in isolated perfused rat livers was not found in isolated perfused guinea pig livers.5. Partially purified pyruvate carboxylases from rat and guinea pig liver do not differ with respect to the apparent K , values for pyruvate and ATP, and also the apparent K , values for acetyl-S-CoA a t different pH values. At constant concentrations of MgATP2-or MnATP2-the pH-optima for the guinea pig enzyme are significantly lower.6. 5-Methoxyindole-2-carbonic acid inhibits gluconeogenesis in guinea pig liver in the same way as in rat liver. Quinolinate, on the other hand is far less inhibitory in guinea pig liver and does not inhibit a t all in isolated perfused pigeon livers.7. Major species differences are found when the activities of the rate limiting enzymes involved in gluconeogenesis are measured under V,,, conditions. Pyruvate carboxylase activity is in the same range in rat and guinea pig liver; pyruvate kinase activity is considerably lower, phosphoenolpyruvate carboxykinase activity is considerably higher in guinea pig liver. According to our results gluconeogenesis in guinea pig liver from lactate or pyruvate is mainly regulated by the concentration of pyruvate.The difference in the regulation of gluconeogenesis between rat and guinea pig liver probably results from a difference in the compartmentalization of phosphoenolpyruvate carboxykinase. A higher rate of futile cycling between phosphoenolpyruvate and pyruvate in rat liver, due to the higher activity of pyruvate kinase in relation to the activities of phosphoenolpyruvate carboxykinase and pyruvate carboxylase, may also cont...