Two plasma membrane proteins, the Na+/Ca2+ exchanger (NCX) and the Ca2+-ATPase, are major regulators of free intraneuronal Ca2+ levels as they are responsible for extrusion of Ca2+ from the intracellular to the extracellular medium. Because disruption of cellular Ca2+ regulation plays a role in damage occurring under conditions of oxidative stress, studies were conducted to assess the sensitivity of the NCX to reactive oxygen species (ROS). Exchanger activity in brain synaptic plasma membranes and in transfected CHO-K1 cells was inhibited following brief exposure to the peroxyl radical generating azo initiator 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) and to peroxynitrite. Incubation with hydrogen peroxide did not alter NCX activity, even at 800 microM concentration. In CHO-K1 cells transiently transfected with the NCX1 isoform of the exchanger, AAPH treatment decreased the maximal transport capacity (Vmax), whereas the K(act) remained unchanged. Peroxynitrite led to an increase in K(act) with no change in Vmax. Loss of activity following exposure to either AAPH or peroxynitrite was associated with the formation of high molecular weight aggregates of NCX, and AAPH also caused fragmentation of the exchanger protein. These findings suggest that the NCX is sensitive to biologically relevant ROS and could be involved in the loss of Ca2+ homeostasis observed under oxidative stress.