Glucose transport across the plasma membrane of mammalian cells is mediated by a family of homologous proteins. Each glucose transporter isoform has a specific tissue distribution which relates to that tissue's demand for glucose. The beta-cells of pancreatic islets are known to express a distinct glucose transporter isoform, termed GLUT 2, which has a high Km for glucose. In this study, we examined the glucose transporter content of normal rat islets and three beta cell lines, beta-TC, HIT and RIN cells. We show that at the protein level, GLUT 2 is the only detectable transporter isoform in normal islets, and that all three cell lines also express detectable GLUT 2. In contrast, all three cell lines expressed high levels of GLUT 1, but this isoform was not detected in normal islets. Neither the native islets nor any of the cell lines expressed GLUT 3. The insulin-responsive glucose transporter GLUT 4 was detected at very low levels in beta-TC cells; to our knowledge, this is the only non-muscle or adipose cell line which expresses this isoform. We propose that the elevated level of GLUT 1 expression, together with a reduced expression of the high Km transporter GLUT 2, may account for the characteristic aberrant patterns of glucose-stimulated insulin release in cell lines derived from beta-cells.