Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular "traffic sign" for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.