The subcellular distribution of glucose transporters in rat hepatocytes and HepG2 cells was studied in the absence and in the presence of insulin. Glucose transporters were quantitated by measuring glucose‐sensitive cytochalasin B binding and by protein immunoblotting using isoform‐specific antibodies. Plasma membrane contamination into subcellular fractions was assessed by measuring distribution of 5′‐nucleotidase and cell surface carbohydrate label. In hepatocytes, GLUT‐2 occurred in a low‐density microsomal (LDM) fraction at a significant concentration, and as much as 15% of cellular GLUT‐2 was found intracellularly that cannot be accounted for by plasma membrane contamination. In HepG2 cells which express GLUT‐1 and GLUT‐2, the two isoforms showed distinct subcellular distribution patterns: GLUT‐2 was highly concentrated in LDM while very little GLUT‐1 was found in this fraction, indicating that a large portion of GLUT‐2 occurs in intracellular organelles. Insulin treatment did not change the subcellular distribution patterns of glucose transporters in both cell types. Our results suggest that rat hepatocytes and HepG2 cells possess an intracellular storage pool for GLUT‐2, but lack the insulin‐responsive glucose transporter translocation mechanism. © 1992 Wiley‐Liss, Inc.