Decreased GLUT4 expression, impaired insulin receptor (IR), IRS‐1, and pp60/IRS‐3 tyrosine phosphorylation are characteristics of adi‐pocytes from insulin‐resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/–)) as a novel model of diabetes to study the development of insulin signaling defects in adipo‐cytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/–) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin‐stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin‐stimulated tyrosine phosphorylation of IR, IRS‐1, and pp60/IRS‐3 were noted with no changes in the cellular content of IR, IRS‐1, and p85 in N/H adipocytes. Insulin‐stimulated protein tyrosine phos‐phorylation was further decreased to 12–23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin‐stimulated, IRS‐1‐associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/–) adipocytes. However, total insulin‐stimulated PI3 kinase activity was normal in H/H GLUT4(+/–) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hypergly‐cemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate‐limiting factor in mediating maximal insulinstimulated glucose uptake in GLUT4(+/–) adipocytes.—Li, J., Houseknecht, K. L., Stenbit, A. E., Katz, E. B., Charron, M. J. Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice. FASEB J. 14, 1117–1125 (2000)