Neurotransmitter and hormone release requires the fusion of secretory vesicles with the plasma membrane of neurons and neuroendocrine cells (i.e. exocytosis). Exocytosis begins with the formation of a fusion pore, an aqueous channel between the vesicle and the plasma membrane through which cargo molecules diffuse out of the vesicle lumen to the cell exterior. After the fusion pore formation it either closes and allows the vesicle to be reused in the next round of exocytosis (“kiss-and-run” exocytosis) or it fully opens leading to the complete merge of vesicle membrane with the plasma membrane (full fusion exocytosis). Not only in stimulated but also in resting synapses and neuroendocrine cells, vesicle cargo appears to be released. For decades, stimulated and spontaneous exocytosis was thought to exhibit similar properties at elementary level, differing only in the probability of occurrence (1). However, recent studies indicate that spontaneous exocytosis differs from the stimulated one in many respects, including in distinct protein requirements for vesicle trafficking, fusion, recycling, and the kinetics of vesicle content discharge. Stimulated hormone discharge from a single lactotroph vesicle of the anterior pituitary, is some 10 to 20 times faster than spontaneous hormone discharge (2), indicating differences in the fusion pore properties in resting and stimulated conditions, respectively. In particular, the fluorescent peptide hormone discharge was monitored with confocal microscopy and compared with the simultaneous loading of vesicle by FM styryl dye. In stimulated vesicles FM 4-64 loading and hormone release occurred within seconds. In contrast, in 50% of spontaneously releasing vesicles, the vesicle content release and the FM 4-64 loading were slow (~3 min). Membrane capacitance measurements revealed regular repetitive transient fusion pore openings (“the pulsing pore”), suggesting that flickering activity of the fusion pore may be the constraint that causes slow vesicle content discharge in resting neuroendocrine cells (2). To see whether the slow release at rest observed by Stenovec et al. (2004) reflects also a relatively narrow fusion pore, we performed additional optical and electrophysiological studies on resting and stimulated pituitary lactotrophs. Kiss-and-run exocytosis, consisting of reversible fusion between the vesicle membrane and the plasma membrane, is considered to lead to full fusion upon stimulation of vesicles containing classical transmitters (3,4). Whether this is also the case in the fusion of peptidergic vesicles is unknown. We analyzed the permeation of FM 4-64 dye and HEPES molecules through spontaneously forming fusion pores in lactotroph vesicles expressing synaptopHluorin, a pH-dependent fluorescent fusion marker (5). Confocal imaging showed that half of the spontaneous exocytotic events exhibited fusion pore openings associated with a change in synaptopHluorin fluorescence, but were impermeable to FM 4-64 (diameter = ~1 nm) and HEPES (diameter = ~0.5 nm). Together with the results obtained by membrane capacitance measurements these findings indicate an open fusion pore diameter 70% of exocytotic events exhibited a larger, FM 4-64–permeable pore (>1 nm). Interestingly, membrane capacitance measurements showed that the majority of exocytotic events in spontaneous and stimulated conditions were transient. However, stimulation increased the frequency of transient events and their fusion pore dwell-time, but decreased the fraction of events with lowest measurable fusion pore. Our results obtained by confocal imaging and membrane capacitance measurements show that kiss-and-run is the predominant mode of exocytosis in resting and in stimulated peptidergic vesicles. Furthermore, our study reveals at the single-vesicle level that stimuli prolong the effective fusion pore opening, which expands from its resting subnanometer diameter (1 nm). Although these findings support the view that spontaneous fusion of peptidergic vesicles is “release-unproductive”, the nature of an energetically stable transient fusion pore and how this relates to membrane trafficking in eukaryotic cells remains to be investigated.