Protein-tyrosine phosphatases (PTPases), in particular PTP1B, have been shown to modulate insulin signal transduction in liver and skeletal muscle in animal models; however, their role in human adipose tissue remains unclear. The uptake of (14)C-D-glucose in response to 10 or 100 nmol/L insulin was measured in isolated subcutaneous adipocytes from subjects with a mean age of 44 years (range, 26 to 58) and mean body mass index (BMI) of 35.6 (range, 29.7 to 45.5). The endogenous activity of total PTPases and specifically of PTP1B in immunoprecipitates was measured in cell lysates under an inert atmosphere with and without added reducing agents. Using nonlinear regression analysis, higher BMI was significantly correlated with lower adipocyte glucose uptake (r = 0.73, P =.01) and with increased endogenous total PTPase activity (r = 0.64, P =.04). Correlation with waist circumference gave similar results. The endogenous total PTPase activity also strongly correlated with insulin-stimulated glucose uptake (R =.89, P <.0001); however, the activity of PTP1B was unrelated to the level of glucose uptake. Consistent with the insulin-stimulated oxidative inhibition of thiol-dependent PTPases reported for 3T3-L1 adipocytes and hepatoma cells, treatment of human adipocytes with 100 nmol/L insulin for 5 minutes lowered endogenous PTPase activity to 37% of control (P <.001), which was increased 25% by subsequent treatment with dithiothreitol in vitro. Cellular treatment with diphenyleneiodonium (DPI), an NADPH oxidase inhibitor that blocks the cellular generation of H(2)O(2) and reduces the insulin-induced reduction of cellular PTPase activity, also diminished insulin-stimulated glucose uptake by 82% (P =.001). These data suggest that total cellular PTPase activity, but not the activity of PTP1B, is higher in more obese subjects and is negatively associated with insulin-stimulated glucose transport. The insulin-stimulated oxidative inhibition of PTPases may also have an important permissive role in the transmission of the insulin signal to glucose transport in human adipocytes.