A glucose binding fraction was obtained by sucrose gradient centrifugation and Sephadex G-200 chromatopgraphy from confluent normal cells and Rous sarcoma virus-transformed fibroblasts. It was more or less loosely bound to the membrane fraction, most strongly in sarcoma cells, and most loosely in slowly growing confluent fibroblasts. In an average of three determinations, the content of binding factor was increased 2.5-fold in transformed cells, and compared reasonably well to a nearly 4-fold increase in glucose uptake. The addition of 5 mug/ml of purified glucose binding factor to the overlaying fluid in the 100-mm plates increased 7-flod the low glucose uptake of starved fibroblasts. The stimulation was an additive increment to the known stimulation by calf serum.