Ca(2+)-dependent activator proteins of secretion (CAPS) 1 and 2 are essential regulators of synaptic vesicle and large dense core vesicle priming in mammalian neurons and neuroendocrine cells. CAPS1 appears to have an additional and as yet unexplained function in vesicular catecholamine uptake or storage as CAPS1-deficient chromaffin cells exhibit strongly reduced vesicular catecholamine levels. Here we describe a role of CAPS proteins in vesicular monoamine uptake. Both CAPS1 and CAPS2 promote monoamine uptake and storage mediated by the vesicular monoamine transporters VMAT1 and VMAT2. Monoamine uptake of vesicular preparations from embryonic brains of CAPS1 deletion mutants is decreased as compared with corresponding preparations from wild type littermates, and anti-CAPS1 or anti-CAPS2 antibodies inhibit monoamine sequestration by synaptic vesicles from adult mouse brain. In addition, overexpression of CAPS1 or CAPS2 enhances vesicular monoamine uptake in Chinese hamster ovary cells that stably express VMAT1 or VMAT2. CAPS function has been linked to the heterotrimeric GTPase G(o), which modulates vesicular monoamine uptake. We found that the expression of CAPS1 is decreased in brain membrane preparations from mice lacking G(o2)alpha, which may explain the reduced monoamine uptake by G(o2)alpha-deficient synaptic vesicles. Accordingly, anti-CAPS1 antibodies do not further reduce monoamine uptake by G(o2)alpha-deficient synaptic vesicles, whereas antibodies directed against CAPS2, whose expression is not altered in G(o2)alpha-deficient brain, still reduce monoamine uptake into G(o2)alpha-deficient vesicles. We conclude that CAPS proteins are involved in optimizing vesicular monoamine uptake and storage mediated by VMAT1 and VMAT2.