We have tested the hypothesis that changes in the levels and cellular location of protein kinase C (PKC) isozymes might be associated with the development of insulin resistance in skeletal muscles from the highfat–fed rat. Lipid measurements showed that triglyceride and diacylglycerol, an activator of PKC, were elevated four- and twofold, respectively. PKC activity assays indicated that the proportion of membraneassociated calcium-independent PKC was also increased. As determined by immunoblotting, total (particulate plus cytosolic) PKC α, ε, and ζ levels were not different between control and fat-fed rats. However, the ratio of particulate to cytosolic PKC ε in red muscles from fat-fed rats was increased nearly sixfold, suggesting chronic activation. In contrast, the amount of cytosolic PKC θ was downregulated to 45% of control, while the ratio of particulate to cytosolic levels increased, suggesting a combination of chronic activation and downregulation. Interestingly, while insulin infusion in glucose-clamped rats increased the proportion of PKC θ in the particulate fraction of red muscle, this was potentiated by fat-feeding, suggesting that the translocation is a consequence of altered lipid flux rather than a proximal event in insulin signaling. PKC ε and θ measurements from individual rats correlated with triglyceride content of red gastrocnemius muscle; they did not correlate with plasma glucose, which was not elevated in fat-fed rats, suggesting that they were not simply a consequence of hyperglycemia. Our results suggest that these specific alterations in PKC ε and PKC θ might contribute to the link between increased lipid availability and muscle insulin resistance previously described using high-fat–fed rats.