While vesicle transport is one of the principal functions of myosin motors in neurones, the role played by specific myosin subtypes in discrete vesicle trafficking is poorly understood. We conducted electrophysiological and morphological experiments to determine whether myosin isoforms II and V might be involved in the transport of small synaptic vesicles in presynaptic nerve terminals of a model cholinergic synapse. Electron microscopy revealed the presence of normal synaptic architecture and synaptic vesicle density in presynaptic terminals of cultured superior cervical ganglion neurones (SCGNs) from myosin Va null rats (dilute‐opisthotonus, dop). Similarly, electrophysiological analyses of synaptic transmission and synaptic vesicle cycling at paired SCGN synapses failed to uncover any significant differences in synaptic development and function between normal and dop rats. Immunocytochemistry and in situ localization of green fluorescent protein (GFP)‐fusion proteins in wild‐type synapses revealed that myosins IIB and Va were distributed throughout the cell soma and processes of SCGNs, while myosins IIA and Vb were not detected in SCGNs. Myosin Va was conspicuously absent in presynaptic nerve terminals, but myosin IIB alone was found to be expressed. Furthermore, synaptic transmission was inhibited by introduction of myosin IIB heavy chain fragments into presynaptic terminals of SCGNs. Together these results suggest that only myosin IIB isoform participates in vesicle trafficking in presynaptic nerve terminals of cultured SCGNs.