The involvement of copper in the pathophysiology of neurodegeneration has been well documented but is not fully understood. Commonly, the effects are attributed to increased reactive oxygen species (ROS) production due to inherent redox properties of copper ions. Here we show copper can have physiological effects distinct from direct ROS production. First, we show that extragranular free copper inhibits the vesicular H(+)-ATPase of resealed chromaffin granule ghosts. Extragranular ascorbate potentiates this inhibition. The inhibition is mixed type with K(is) = 6.8 +/- 2.8 micromol/L and K(ii) = 3.8 +/- 0.6 micromol/L, with respect to ATP. Second, extracellular copper causes an inhibition of the generation of a pH-gradient and rapid dissipation of pre-generated pH and catecholamine gradients. Copper chelators and the ss-amyloid peptide 1-42 were found to effectively prevent the inhibition. The inhibition is reversible and time-independent suggesting the effects of extracellular copper on H(+)-ATPase is direct, and not due to ROS. The physiological significance of these observations was shown by the demonstration that extracellular copper causes a dramatic perturbation of dopamine metabolism in SH-SY5Y cells. Thus, we propose that the direct inhibition of the vesicular H(+)-ATPase may also contribute to the neurotoxic effects of copper.