Synapsin I is a neuron-specific phosphoprotein primarily localized at the presynaptic terminals, where it is thought to play an important role in the mechanisms involved in neurotransmitter release. Its interaction with cytoskeletal proteins and with small synaptic vesicles is regulated in vitro by phosphorylation by a calcium/calmodulin-dependent kinase. Here, we present the first evidence that, in the mouse retinal ganglion cells, synapsin I, moving along the axon with the slow component of axonal transport, is phosphorylated in vivo at both the head and tail regions. In addition, our data suggest that, after synapsin I has reached the nerve endings, the relative proportion of differently phosphorylated molecules of synapsin I changes, and that these changes lead to a decrease of the overall content of phosphorus. The more basic forms, here collectively referred to as beta-forms, become predominant at the terminals after 7 d postlabeling, when the bulk of transported synapsin I has entered the superior colliculus. Along the axon, phosphorylation could be functional in preventing synapsin I from forming, with actin, a dense meshwork that would restrict organelle movement. On the other hand, at the terminals, the dephosphorylation-phosphorylation of synapsin I may regulate the clustering of small synaptic vesicles and modulate neurotransmitter release by controlling the availability of small synaptic vesicles for exocytosis.