In this study we determined whether alterations in the expression of GLUT4, the major insulin-regulatable glucose transporter, in skeletal muscle could explain the insulin-resistant glucose uptake characteristic of both dietary-induced and genetic obesity. GLUT1 expression was measured for comparison. To assess glucose transporter protein levels in dietary-induced obesity, postnuclear membranes were prepared from hindlimb muscle of Sprague-Dawley rats fed chow ad libitum (control), a high calorie/carbohydrate diet, or a high fat (80%) diet for 7 weeks. Immunoblotting revealed that GLUT4 protein levels decreased 34% in high fat-fed rats, but were unaltered in high calorie/carbohydrate-fed obese rats compared to control values. GLUT4 mRNA per DNA decreased 47% in muscle of high fat-fed rats compared to that in control or high calorie-fed rats; GLUT1 mRNA was reduced 31%, and actin mRNA tended to decrease (29%). To assess GLUT4 and GLUT1 expression in genetic obesity, similar studies were carried out in 5- and 20-week-old lean and obese Zucker rats as well as in 20-week-old obese Zucker rats 36 h after streptozotocin injection to lower insulin levels. GLUT4 protein and mRNA levels were unaltered in hindlimb muscle of obese Zucker rats at either age or in the acutely diabetic state, whereas GLUT1 protein and mRNA levels decreased 40-45%. Comparison of these results with recent data in adipocytes demonstrates tissue-specific regulation of expression of GLUT4 and GLUT1. Thus, obesity due to high fat feeding, but not that due to high calorie/carbohydrate feeding or genetics, is associated with pretranslational suppression of GLUT4 expression in skeletal muscle. In at least some forms of obesity, the level of GLUT4 expression in muscle appears to be only one factor in, or may even be unrelated to, the degree of insulin-responsive glucose transport in vivo.