The properties and subcellular distribution of cysteine string proteins (csps) were analyzed in peptidergic nerve terminals of the rat neurohypophysis. Polyclonal antibodies raised against recombinant rat brain csp recognized a 36 kDa protein in isolated neurosecretosomes from the post-pituitary. After chemical deacylation, a single 27 kDa form was detected that displayed identical properties to csps in a whole-brain synaptosomal fraction. Immunoisolation demonstrated that synaptophysin and csps were located in the same vesicles. Density gradient centrifugation of postsynaptosomal supernatants of neurohypophysial homogenates revealed that csps and VAMP were present in two distinct vesicle populations. Synaptophysin was only detected in the slowly migrating population corresponding to small synaptic vesicles, whereas arginine vasopressin was present in the more rapidly sedimenting population indicating that it contains large dense core vesicles (LDCVs). Immobilized antibodies against csp, synaptotagmin, or VAMP captured vesicular arginine vasopressin confirming the association of these proteins with LDCVs. Co-immunoprecipitation assays with proteins solubilized from neurohypophysial or whole-brain nerve terminals failed to reveal complexes containing csp and [125I]omegaGVIA receptors. These results indicate that csps in the CNS are associated with both small synaptic vesicles and LDCVs. However, they do not provide support for the hypothesis that protein complexes implicated in exocytosis, which interact with presynaptic N-type calcium channels, contain csps.