Non enzymatic glycation could be involved in the early impairment of Na+/K+ ATPase that occurs in sciatic nerve of diabetic rats. In fact, decrease of Na+/K+ ATPase activity is one of the first alterations showed in experimental diabetic neuropathy. In this respect, it is known that in the presence of transition metals under physiological conditions, glucose can autoxidize yielding hydrogen peroxide (H2O2) and free radical intermediates, which, in turn, inhibit the cation pump. Our experiments were designed to determine if glucose autoxidation has any relevance in the early steps of Na+/K+ ATPase experimental glycation. Compared experiments with and without the sodium borohydride (NaBH4) reduction step demonstrated that incubation of brain Na+/K+ ATPase with glucose 6-phosphate (G 6-P) and trace metals induced a significant decrease in enzyme activity dramatically enhanced by addition of copper (Cu2+). A concomitant production of H2O2 was noticed. The presence of diethylenetriaminepentaacetic acid (DTPA), a strong metal chelator, completely prevented Na+/K+ ATPase impairment and hydrogen-peroxide formation. No gross structural and conformational alterations of the enzyme can be demonstrated by intrinsic and extrinsic fluorescence measurements. Our results suggest that during the exposure of brain NA+/K+ ATPase to glucose 6-phosphate in vitro (experimental glycation), the decrease in activity can be correlated, at lease in the early phases, to metal-catalyzed production of oxidative species, such as H2O2, through the glucose autoxidation process, and not to glucose attachment to the enzyme. Since plasma hydroperoxides and copper appear to be elevated in diabetic patients with complications, our data suggest a critical role for oxidative reactions in the pathophysiology of the chronic complications of diabetes like neuropathy.