Purpose: To investigate the mechanisms related to F-18-FDG uptake by tumors, F-18-FDG accumulation was compared with glucose transporter-1 (Glut-1) expression and hexokinase activity in various human cancer cell lines. Materials and Methods: Human colon cancer (SNU-C2A, SNU-C4, SNU-C5), hepatocellular carcinoma (SNU-387, SNU-423, SNU-449), lung cancer (NCI-H522, NCI-H358, NCI- H1299), uterine cervical cancer (HeLa, HeLa 229, HeLa S3) and brain tumor (A172, Hs 683) cell lines were used. After 24 hr incubation of 5×105 cells, 37 kBq F-18-FDG was added and the uptake by cells at 10 min was measured using a gamma counter. Hexokinase activity was measured by continuous spectrophotometric rate determination. To measure mitochondrial hexokinase activity, mitochondrial fraction was separated by a high speed centrifuge. Immunohistochemical staining of Glut-1 was performed, and graded as 0, 1, 2, or 3 according to expression. Results: There was difference among F-18-FDG uptake, total and mitochondrial hexokinase activity, and Glut-1 expression with different cancer cell lines. The correlations of F-18-FDG with total hexokinase and mitochondrial hexokinase activity were low (r=0.27 and 0.26, respectively). Glut-1 expression showed a good correlation with F-18-FDG uptake (ρ=0.81, p=0.0015). Previously, we reported no correlation of F-18-FDG uptake with hexokinase activity in colon cancer cell lines. Thus, when colon cancer cells were excluded, F-18-FDG uptake showed higher correlation with total hexokinase and mitochondrial hexokinase activity (r=0.81, p=0.0027 and r=0.81, p=0.0049, respectively). Conclusion: Both Glut-1 expression and hexokinase activity were contributing factors related to F-18-FDG accumulation in human cancer cell lines. The relative contribution of Glut-1 expression and hexokinase activity, however, was different among different cancer cell types. (Korean J Nucl Med 2000;34:294-302)