The isolated chick ciliary neuron calyx synapse preparation was used to test cysteine string protein (CSP) action on presynaptic N-type Ca2+ channels. Endogenous CSP was localized primarily to secretory vesicle clusters in the presynaptic nerve terminal. Introduction of recombinant CSP into the voltage clamped terminal resulted in a prominent increase in Ca2+ current amplitude. However, this increase could not be attributed to a change in Ca2+ channel kinetics, voltage dependence, prepulse inactivation, or G protein inhibition but was attributed to the recruitment of dormant channels. Secretory vesicle associated endogenous CSP may play an important role in enhancing Ca2+ channel activity at the transmitter release site.