The effects of tumor necrosis factor-α (TNF) on glutamine GLN transport by cultured human fibroblasts were studied. Uptake of 3H-GLN was assayed in both the presence and absence of sodium in order to differentiate Na+-dependent and Na+-independent transport systems. GLN transport was linear (r = 0.99) for at least 15 min and occurred predominantly via a single Na+-dependent pathway, consistent with System ASC. Incubation of fibroblasts with TNF (1000 units/ml) for 12 hr resulted in a significant decrease in system ASC-mediated glutamine transport activity. TNF did not alter cell morphology or protein content. Kinetic studies indicated that the decrease in carrier-mediated Na+-dependent GLN transport was not due to a change in transporter affinity (Km = 117 ± 23 μM in controls vs 86 ± 23 μM in TNF, P = NS), but instead to a 45% decrease in maximal transport rate (Vmax = 4088 ± 354 pmole/mg protein/30 sec in controls vs 2230 ± 510 in TNF, P < 0.05). TNF also decreased Na+-independent transport by 50% (mean uptake of 50 μM GLN = 94 ± 13 pmole/mg protein/30 sec in controls vs 46 ± 6 in TNF, P < 0.02). In human fibroblasts, the activity of System ASC, which has generally been viewed as a hormonally unresponsive carrier, is decreased by TNF. This impairment in glutamine transport may result in inadequate amounts of intracellular glutamine to support fibroblast metabolism and possibly function. © 1992.