The dynamics of non-esterified fatty acid (NEFA) metabolism in lactating dairy cows requires quantification if we are to understand how dietary treatments and disease influence changes in body condition (adipose reserves) and the production of milk fat. We present here a novel compartmental model that employs the pattern of plasma glucose concentrations to predict the dynamic changes that occur in plasma NEFA concentrations during an intravenous glucose tolerance test (IVGTT) in lactating dairy cows. The model was developed using data obtained from ten early-lactation, Holstein-Friesian cows given a standard IVGTT. The model described all of the major features of the NEFA response to an IVGTT; it was consistent with physiological processes and provided a number of parameters that can be used to quantify NEFA production and utilization. For all of the individual cows, all model parameters were well identified and usually had CV<10% of their estimated values. In the model, elevated plasma glucose concentrations cause an increase in the level of glucose in a remote compartment, which in turn suppresses the rate of entry of NEFA to the plasma compartment. The means (±sd) for the five adjustable parameters of the model were: rate of entry of NEFA to the plasma pool (SFFA) 183±71 [μmol l−1 min−1], rate of removal (oxidation, sequestration in adipose tissue and uptake by the mammary gland for milk production) of NEFA from the plasma pool (KFFA) 0·140±0·047 [min−1], a threshold parameter (gs) representing a plasma glucose concentration above which elevated levels of plasma glucose result in entry of glucose into a ‘remote’ or inaccessible glucose compartment, 3·30±0·52 [mmol/l], a rate constant (K) describing the movement of plasma glucose (above gs) into a remote compartment 0·063±0·033 [min−1] and a parameter Φ which is a Michaelis Menten type affinity constant which modulates the extent to which remote glucose inhibits the provision of NEFA to the plasma pool, 0·812±0·276 [mmol/l]. It is concluded that the model is suitable to describe NEFA kinetics in lactating dairy cows and it may have application in other species.