A key step during neurotransmitter or hormone release (exocytosis) is the formation of a nanometer-sized fusion pore that connects the plasma membrane to the synaptic or secretory vesicle. The pore can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics affect the amount and kinetics of released cargo, and vesicle recycling. However, factors regulating pore dynamics are poorly understood, in large part due to a lack of biochemically defined assays that can probe single, nanometer-sized pores. We have developed a novel assay that probes single fusion pores formed between 15-30 nm flat lipid bilayer nanodiscs reconstituted with neuronal/exocytotic vesicular soluble v-SNARE proteins and cells engineered to express cognate “flipped” target membrane t-SNAREs on their surfaces, with the SNARE domain facing the extracellular medium. Formation of a complex between the v- and t-SNAREs leads to fusion of the nanodisc and cell membranes with the apearance of a pore connecting the cell cytosol to the exterior. Using cell-attached, voltage-clamped measurements, currents passing through single pores can be detected and used to monitor pore properties.