A novel class of intracellular chloride channels, the p64 family, has been found on several types of vesicles. These channels, acting in concert with the electrogenic proton pump, regulate the pH of the vesicle interior, which is critical for vesicular function. Here we describe the molecular cloning of p64H1, a p64 homolog, from both human and cow. Northern blot analysis showed that p64H1 is expressed abundantly in brain and retina, whereas the other members of this family (e.g., p64 and NCC27) are expressed only at low levels in these tissues. Immunohistochemical analysis of p64H1 in rat brain, using an affinity-purified antibody, revealed a high level of expression in the limbic system-the hippocampal formation, the amygdala, the hypothalamus, and the septum. Immunoelectron microscopic analysis of p64H1 in hippocampal neurons demonstrated a striking association between p64H1 and large dense-core vesicles (LDCVs) and microtubules. In contrast, very low p64H1 labeling was found in perikarya or associated with small synaptic vesicles (SSVs) in axonal profiles. Immunoblot analysis confirmed that p64H1 is colocalized with heavy membrane fractions containing LDCVs rather than the fractions containing SSVs. These results suggest that p64H1-mediated Cl- permeability may be involved in the maintenance of low internal pH in LDCVs and in the maturation of LDCVs and the biogenesis of functional neuropeptides.