Since the fusion pore of the secretory vesicle is resealed before complete dilation during 'kiss-and-run' exocytosis, their cargoes are not completely released. Although the transient fusion pore is kept open for several seconds, the precise mechanisms that control fusion pore maintenance, and their physiological significance, are not well understood. Using dual-colour TIRF (total internal reflection fluorescence) microscopy in neuroendocrine PC12 cells, we show that myosin II regulates the fusion pore dynamics during kiss-and-run exocytosis. The release kinetics of mRFP (monomeric red fluorescent protein)-tagged tPA (tissue plasminogen activator) and Venus-tagged BDNF (brain-derived neurotrophic factor), which show slower release kinetics than NPY (neuropeptide Y)-mRFP and insulin-mRFP, were prolonged by the overexpression of a wild-type form of the RLC (myosin II regulatory light chain). In contrast, overexpression of a dominant-negative form of RLC shortened the release kinetics. Using spH (synapto-pHluorin), a green fluorescent protein-based pH sensor inside the vesicles, we confirmed that the modulation of the release kinetics by myosin II is due to changes in the duration of fusion pore opening. In addition, we revealed that the amount of hormone released into the extracellular space upon stimulation was increased by overexpression of wild-type RLC. We propose that the duration of fusion pore opening is regulated by myosin II to control the amount of hormone released from a single vesicle.