Epidermal growth factor (EGF) is known to enhance wound healing, partly by stimulating fibroblast proliferation. We studied the effects of EGF on the cell membrane transport of glutamine (GLN) and glucose, the two major fuels utilized by fibroblasts. Human foreskin fibroblasts were grown to confluence on culture wells in Dulbecco's MEM. EGF (10 ng/ml) or vehicle was added to the cells; substrate uptake was assayed at various timepoints. As previously shown, GLN uptake was predominantly Na+-dependent and occurred via two distinct carriers, the low-affinity System A and the high-affinity System ASC. Activity of the high affinity system was assayed with [3H]GLN while blocking the low-affinity system with excess α-methylaminoisobutyric acid (MeAIB), a selective System A substrate. [3H]-MeAIB uptake was used to assay activity of the low affinity carrier. EGF caused a time-dependent increase in GLN transport by fibroblasts. The activity of the low-affinity GLN carrier (System A) increased by 60% after 18 hr of exposure to EGF. The increase in the high-affinity agency revealed an early increase at 1-2 hr and a late increase at 18-24 hr. The enhanced transport activity was due to an increase in V(max) with no change in carrier affinity. In contrast, glucose uptake was Na+-independent and EGF caused a decrease in glucose uptake at early time points. EGF treatment results in divergent regulation of glutamine and glucose transport in human fibroblasts. This response may be designed to provide GLN for both energy production and nucleotide biosynthesis to support cell metabolism and proliferation. © 1993 Academic Press. All rights reserved.