To directly assess the effects of the biguanide, metformin, on hepatic gluconeogenesis, it was added at high therapeutic levels (90 microg/ml) to the medium perfusing an isolated rat liver. Lactate (1 mg/min) was infused simultaneously along with [14C]lactate with or without [3H]lactate. [6-(3)H]glucose was added at the beginning of the perfusion in studies where [3H]lactate was not infused. Glucose levels decreased relative to control studies (metformin dose = 0) and lactate concentrations increased in this closed system. Quantitative analysis of the relationship between labeled glucose and lactate indicated that the flux of carbon from lactate to glucose and CO2 was halved, whereas reflux from glucose to lactate increased by approximately 80%. This was corroborated by measurement of labeled lactate extraction as well as glucose, CO2, and lactate production across the liver. Glycogen content of the liver fell by 60% relative to control and was greater for the gluconeogenic pathway. These data are consistent with an inhibitory action of metformin on gluconeogenesis, which is due to a primary inhibition of hepatic lactate uptake.