Euglycemic, hyperinsulinemic clamp tests have shown that adult ruminants are less insulin-sensitive than monogastric omnivores. The present study was carried out to elucidate possible cellular mechanisms contributing to this impaired insulin sensitivity of ruminants. Western blotting was used to measure the glucose transporter 1 and 4 (GLUT1, GLUT4) of oxidative (musculus masseter, diaphragm) and glycolytic (musculus longissimus dorsi, semitendinosus) skeletal muscle crude membranes of pigs and cows. Muscles were characterized biochemically. To determine insulin-stimulated 3-O-methylglucose uptake and GLUT4 translocation, porcine and bovine musculus semitendinosus strips were removed by open muscle biopsy (cows: epidural anesthesia, pigs: azaperone/ketamine narcosis + epidural anesthesia), and incubated without and with 0.1 or 20 mIU/ml insulin. GLUT4 translocation was analyzed using subcellular fractionation techniques to isolate partially purified plasma membranes and cytoplasmatic vesicles, and by Western blotting. GLUT4 protein contents were significantly higher in oxidative than in glycolytic muscles in pigs and cows. GLUT1 protein contents were significantly higher in glycolytic than in oxidative muscles in bovines but not in porcines. The 3-O-methylglucose uptake into musculus semitendinosus was similar in both species. Maximum insulin-induced GLUT4 translocation into musculus semitendinosus plasma membrane was significantly lower in bovines than in porcines. These results indicate that GLUT1 is the predominant glucose transporter in bovine glycolytic muscles and that a reinforced insulin-independent glucose uptake via GLUT1 may compensate for the impaired insulin-stimulated GLUT4 translocation, resulting in a similar 3-O-methylglucose uptake in bovine and porcine musculus semitendinosus. These findings may explain at least in part the impaired in vivo insulin sensitivity of adult ruminants compared with that of omnivorous monogastric animals.