Oversupply and underutilization of lipid fuels are widely recognized to be strongly associated with insulin resistance in skeletal muscle. Recent attention has focused on the mechanisms underlying this effect, and defects in mitochondrial function have emerged as a potential player in this scheme. Because evidence indicates that lipid oversupply can produce abnormalities in extracellular matrix composition and matrix changes can affect the function of mitochondria, the present study was undertaken to determine whether muscle from insulin-resistant, nondiabetic obese subjects and patients with type 2 diabetes mellitus had increased collagen content. Compared with lean control subjects, obese and type 2 diabetic subjects had reduced muscle glucose uptake (P < 0.01) and decreased insulin stimulation of tyrosine phosphorylation of insulin receptor substrate-1 and its ability to associate with phosphatidylinositol 3-kinase (P < 0.01 and P < 0.05). Because it was assayed by total hydroxyproline content, collagen abundance was increased in muscle from not only type 2 diabetic patients but also nondiabetic obese subjects (0.26 ± 0.05, 0.57 ± 0.18, and 0.67 ± 0.20 μg/mg muscle wet wt, lean controls, obese nondiabetics, and type 2 diabetics, respectively), indicating that hyperglycemia itself could not be responsible for this effect. Immunofluorescence staining of muscle biopsies indicated that there was increased abundance of types I and III collagen. We conclude that changes in the composition of the extracellular matrix are a general characteristic of insulin-resistant muscle.