The psychostimulant, methylphenidate (MPD), is commonly prescribed to treat attention‐deficit hyperactivity disorder. MPD binds to the neuronal dopamine (DA) transporter, where it blocks the inward transport of DA. The present study expands upon these findings by examining the effects of in vivo MPD administration on the vesicular monoamine transporter‐2 (VMAT‐2) in membrane‐associated vesicle and cytoplasmic vesicle subcellular fractions (i.e., those vesicles that do and do not co‐fractionate with synaptosomal membranes after osmotic lysis, respectively) isolated from lysates of rat striatal synaptosomes. The results indicate that a single MPD administration redistributes VMAT‐2 and associated vesicles within nerve terminals away from the synaptosomal membranes and into the cytoplasm, as assessed 1 hour after treatment. DA transport is also increased by MPD in both vesicle fractions (on account of vesicle trafficking in the cytoplasmic vesicles and to kinetic upregulation of the VMAT‐2 in the membrane‐associated vesicles). This, in turn, leads to an increase in the DA content of both vesicle fractions as well as an increase in the velocity and magnitude of K⁺‐stimulated DA release from striatal suspensions. Taken together, these data show that the trafficking, DA sequestration function, DA content, and exocytotic DA release function of synaptic vesicles can all be pharmacologically manipulated by in vivo MPD treatment. These findings may provide important insights useful for understanding and treating disorders involving abnormal DA transmission including drug abuse, Parkinson's disease, and attention‐deficit hyperactivity disorder.