Amphiphysin I (amphI) is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE). Some amphI phosphorylation sites (phosphosites) have been identified with in vitro studies or phosphoproteomic screens. We used a multi-faceted strategy including ³²P-tracking to identify all in vivo amphI phosphosites and determine their relative abundance and potential relevance to SVE. AmphI was extracted from ³²P-labeled synaptosomes, phosphopeptides isolated from proteolytic digests using TiO₂ chromatography and mass spectrometry revealed thirteen sites: Serines-250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, 626, and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src-homology 3 domain. Hierarchical phosphorylation of Ser-262 preceded phosphorylation of Ser-268, 272, 276 and 285. Off-line HPLC separation and 2D tryptic mapping of ³²P labeled amphI revealed that Thr-310, Ser-293, Ser-285, Ser-272, Ser-276 and Ser-268, contained the highest ³²P incorporation and were the most stimulus-sensitive. Individually, Thr-310 and Ser-293 were the most abundant phosphosites, incorporating 16% and 23% of the ³²P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272 and Ser-285 had 27% of the 32P. Evidence for a role for at least one proline-directed protein kinase and one non-proline directed kinase was obtained. Four phosphosites predicted for non-proline directed kinases, Ser-626, 250, 252, and 539 contained low amounts of ³²P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated, since it did not incorporate ³²P during the 1 hour labeling period. Multiple phosphosites from amphI co-migrating synaptosomal proteins were also identified: including SGIP, AAK1, eps15R, MAP6, a/ß-adducin and HCN1. The results reveal two sets of amphI phosphosites that are either dynamically turning-over or constitutively phosphorylated in nerve terminals, and improves understanding of the role of individual amphI sites or phosphosites clusters in synaptic SVE.