Regulation of exocytosis by voltage-gated K+ channels has classically been viewed as inhibition mediated by K+ fluxes. We recently identified a new role for Kv2.1 in facilitating vesicle release from neuroendocrine cells, which is independent of K+ flux. Here, we show that Kv2.1-induced facilitation of release is not restricted to neuroendocrine cells, but also occurs in the somatic-vesicle release from dorsal-root-ganglion neurons and is mediated by direct association of Kv2.1 with syntaxin. We further show in adrenal chromaffin cells that facilitation induced by both wild-type and non-conducting mutant Kv2.1 channels in response to long stimulation persists during successive stimulation, and can be attributed to an increased number of exocytotic events and not to changes in single-spike kinetics. Moreover, rigorous analysis of the pools of released vesicles reveals that Kv2.1 enhances the rate of vesicle recruitment during stimulation with high Ca2+, without affecting the size of the readily releasable vesicle pool. These findings place a voltage-gated K+ channel among the syntaxin-binding proteins that directly regulate pre-fusion steps in exocytosis.