C57BL/6J (B6) and AKR/J (AKR) inbred strains of mice develop a comparable degree of obesity when fed a high-fat diet. However, although obese B6 mice are more glucose intolerant, obese AKR mice are more insulin resistant. To understand the basis for these strain differences, we characterized features of adiposity and glucose homeostasis in mice fed a high-fat diet for 8 weeks. The results indicated that despite hyperglycemia and impaired glucose tolerance, B6 mice have lower plasma insulin and are more insulin sensitive than AKR mice. Compared with adipose tissue of AKR mice, adipose tissue of B6 mice contained about threefold higher levels of total membrane-bound GLUT4 protein, whereas in skeletal muscle the levels were similar. Uptake of 2-[(14)C]deoxyglucose in vivo was reduced by a high-fat diet in adipose tissue, but not in skeletal muscle. Surprisingly, no significant differences in uptake occurred between the strains, despite the differences in GLUT4; however, glucose flux was calculated to be slightly higher in B6 mice. Higher expression of PEPCK in the liver of B6 mice, under both standard-diet and high-fat-diet conditions, suggests a plausible mechanism for elevated glycemia in these mice. In conclusion, phenotypic variation in insulin resistance and glucose production in the B6 and AKR strains could provide a genetic system for the identification of genes controlling glucose homeostasis.