Proteins constituting the presynaptic machinery of vesicle release undergo substantial conformational changes during the process of exocytosis. While changes in the conformation make proteins vulnerable to aggregation and degradation, little is known about synaptic chaperones which counteract these processes. We show that the cell adhesion molecule CHL1 directly interacts with and regulates the activity of the synaptic chaperones Hsc70, CSP and alpha SGT. CHL1, Hsc70, CSP and alpha SGT form predominantly CHL1/Hsc70/alpha SGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and alpha SGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/alpha SGT and CHL1/CSP complexes, respectively, indicating a remarkable selectivity of a presynaptic chaperone activity for proteins of the exocytotic machinery. In mice with genetic ablation of CHL1, chaperone activity in synapses is reduced and the machinery for synaptic vesicle exocytosis and, in particular, the SNARE complex is unable to sustain prolonged synaptic activity. Thus, we reveal a novel role for a cell adhesion molecule in selective activation of the presynaptic chaperone machinery.