Glucose transporter (GLUT) expression and hexokinase activity are thought to be related to high [18F]-fluorodeoxyglucose (FDG) uptake in tumor cells, but their relative importance is still unknown. To determine which is the predominant factor in FDG uptake in tumor cells, cultured tumor cell lines and a normal cell line were studied in vitro with respect to 2-deoxyglucose (DG) uptake, hexokinase activity, and the initial uptake rate of 3-O-methylglucose (3-O-MG) transport, which is generally accepted as indicating the amount of GLUT expressed on the plasma membrane. In 16 types of tumor cells and one fibroblast cell line, DG uptake was assessed for 60 min, the initial uptake rate of 3-O-MG transport was measured for 1 min, and total hexokinase activity, including that in the mitochondrial fraction, was determined. Across all 16 tumor cell lines, there was a significant correlation between DG uptake and 3-O-MG transport (p = 0.0012, F test), but not between DG uptake and hexokinase activity. Hexokinase activity of the tumor cells was comparable to that of the human fibroblast cells in the exponential growth phase. Most tumor cells showed higher DG uptake and 3-O-MG transport than the human fibroblast cells. The results suggest that DG uptake of cultured tumor cells is governed by GLUT expression, which may be a distinct characteristic of the neoplastic process.