Hepatocytes were isolated from sixteen fasting neonatal pigs and used in two experiments: (1) to determine the effect of various factors on the ability for hepatic oxidation of fatty acids and (2) to clarify the relationship between fatty acid oxidation and glucose synthesis. In Expt 1, newborn pigs were either fasted from birth for 24 h or allowed to suck ad lib. for 3 d followed by a 24 h fast. In the presence of pyruvate, oxidation of octanoate (2 mM) was about 30-fold greater than oleate (1 mM) regardless of age, but glucose synthesis was not enhanced beyond that observed for pyruvate alone. Inclusion of carnitine (1 mM), glucagon (100 nM) or dibutryl cAMP (50 μM) in the incubation media did not stimulate either fatty acid oxidation (octanoate or oleate) or glucose synthesis. Extending the period of fasting to 48 h (Expt 2) failed to enhance the fatty acid oxidative capacity or glucose synthesis rate. Likewise, the redox potential of the giuconeogenic substrate (lactate v. pyruvate) did not influence glucose synthesis regardless of the oxidative capacity exhibited for fatty acids. These data indicate that fatty acid oxidative capacity is not the first limiting factor to full expression of gluconeogenesis in hepatocytes isolated from fasted newborn pigs.