Methamphetamine (MA) produces selective degeneration of dopamine (DA) neuron terminals without cell body loss. While excitatory amino acids (EAAs) contribute to MA toxicity, terminal loss is not characteristic of excitotoxic lesions nor is excitotoxicity selective for DA fibers; rather, EAAs may modulate MA-induced DA turnover, suggesting that DA-dependent events play a key role in MA neurotoxicity. To examine this possibility, we used postnatal ventral midbrain DA neuron cultures maintained under continuous EAA blockade. As in vivo, MA caused neurite degeneration but minimal cell death. We found that MA is a vacuologenic weak base that induces swelling of endocytic compartments; MA also induces blebbing of the plasma membrane. However, these morphological changes occurred in MA-treated cultures lacking DA neurons. Therefore, while collapse of endosomal and lysosomal pH gradients and vacuolation may contribute to MA neurotoxicity, this does not explain selective DA terminal degeneration. Alternatively, MA could exert its neurotoxic effects by collapsing synaptic vesicle proton gradients and redistributing DA from synaptic vesicles to the cytoplasm. This could cause the formation of DA-derived free radicals and reactive metabolites. To test whether MA induces oxidative stress within living DA neurons, we used 2,7-dichlorofluorescin diacetate (DCF), an indicator of intracellular hydroperoxide production. MA dramatically increased the number of DCF-labeled cells in ventral midbrain cultures, which contain about 30% DA neurons, but not in nucleus accumbens cultures, which do not contain DA neurons. In the DA neuron cultures, intracellular DDF labeling was localized to axonal varicosities, blebs, and endocytic organelles. These results suggest that MA redistributes DA from the reducing environment within synaptic vesicles to extravesicular oxidizing environments, thus generating oxygen radicals and reactive metabolites within DA neurons that may trigger selective DA terminal loss.