The insulin-sensitive glucose transporter, GLUT4, is the most abundant facilitative glucose transporter in muscle and adipose tissue, the major sites for postprandial glucose disposal. To assess the role of GLUT4 in glucose homeostasis, we have disrupted the murine GLUT4 gene. Because GLUT4 has been shown to be dysregulated in pathological states such as diabetes and obesity, it was expected that genetic ablation of GLUT4 would result in abnormal glucose homeostasis. The mice deficient in GLUT4 (GLUT4-null) are growth-retarded and exhibit decreased longevity associated with cardiac hypertrophy and severely reduced adipose tissue deposits. Blood glucose levels in female GLUT4-null mice are not significantly elevated in either the fasting or fed state; in contrast, male GLUT4-null mice have moderately reduced glycaemias in the fasted state and increased glycaemias in the fed state. However, both female and male GLUT4-null mice exhibit postprandial hyperinsulinaemia, indicating possible insulin resistance. Increased expression of other glucose transporters is observed in the liver (GLUT2) and heart (GLUT1) but not skeletal muscle. Oral glucose tolerance tests show that both female and male GLUT4-null mice clear glucose as efficiently as controls, but insulin tolerance tests indicate that these mice are less sensitive to insulin action. The GLUT4-null mice demonstrate that functional GLUT4 protein is not required for maintaining nearly normal glycaemia but that GLUT4 is absolutely essential for sustained growth, normal cellular glucose and fat metabolism, and expected longevity.