The -subunits of the trimeric Go class of GTPases, comprising the splice variants Go1 and Go2, are abundantly expressed in brain and reside on both plasma membrane and synaptic vesicles. Go2 is involved in the vesicular storage of monoamines but its physiological relevance is still obscure. We now show that genetic depletion of Go2 reduces motor activity induced by dopamine-enhancing drugs like cocaine, as repeated injections of cocaine fail to provoke behavioral sensitization in Go2^-/- mice. In Go2^-/- mice, D1 receptor signaling in the striatum is attenuated due to a reduced expression of Golf and Gs. Following cocaine treatment, Go2^-/- mice have lower D1 and higher D2 receptor amounts compared to wild-type mice. The lack of behavioral sensitization correlates with reduced dopamine levels in the striatum and decreased expression of tyrosine hydroxylase. One reason for the neurochemical changes may be a reduced uptake of monoamines by synaptic vesicles from Go2^-/- mice as a consequence of a lowered set point for filling. We conclude that Go2 optimizes vesicular filling which is instrumental for normal dopamine functioning and for the development of drug-induced behavioral sensitization.—Brunk, I., Blex, C., Sanchis-Segura, C., Sternberg, J., Perreau-Lenz, S., Bilbao, A., Hörtnagl, H., Baron, J., Juranek, J., Laube, G., Birnbaumer, L., Spanagel, R., Ahnert-Hilger, G. Deletion of Go2 abolishes cocaine-induced behavioral sensitization by disturbing the striatal dopamine system.