Initial rates of glucose entry into isolated bovine mammary epithelial cells displays moderate degrees of asymmetry and cooperative interactions between export and import sites. The present study examined the hypothesis that these kinetic features are due to compartmentalization of intracellular glucose. Net uptake of 3-O-methyl-D-[1-³H]glucose (3OMG) by isolated bovine mammary epithelial cells was measured at 37 °C. The time course of 3OMG net uptake was better fitted by a double exponential equation than by a single or triple exponential equation. Compartmental analysis of the time course curve suggested that translocated 3OMG is distributed into two compartments with fractional volumes of 32.6 ± 5.7% and 67.4 ± 5.7%, respectively. The results support the view that glucose transport in bovine mammary epithelial cells is a multi-step process consisting of two serial steps: fast, carrier-mediated, symmetric translocation of sugar across the cell plasma membrane into a small compartment, and subsequent slow exchange of post-translocated sugar between two intracellular compartments. A 3-compartment model of this system successfully simulated the observed time course of 3OMG net uptake and the observed dependence of unidirectional entry rates on intra- and extracellular 3OMG concentrations. Simulations indicated that backflux of radiolabeled sugar from the small compartment to extracellular space during 15 s of incubation gives rise to the apparent asymmetry, trans-stimulation and cooperativity of mammary glucose transport kinetics. The fixed-site carrier model overestimated the role of glucose accumulation in cells and its features can be accounted for by the compartmentalization of intracellular sugar.