We examined the hypothesis that myosin Va, by transporting purinergic vesicles to the varicosity membrane for exocytosis, plays a key role in purinergic vesicular neurotransmission. Studies were performed in wild-type (WT) and myosin Va-deficient dilute, brown, nonagouti (DBA) mice. Intracellular microelectrode recordings were made in mouse antral muscle strips. Purinergic inhibitory junction potential (pIJP) was recorded under nonadrenergic noncholinergic conditions after masking the nitrergic junction potentials. DBA mice showed reduced pIJP but normal hyperpolarizing response to P2Y1 receptor agonist MRS-2365. To investigate the mechanism of reduced purinergic transmission in DBA mice, studies were performed in isolated varicosities obtained from homogenates of whole gut tissues by ultracentrifugation and sucrose cushion purification. Purinergic varicosities were identified in tissue sections and in isolated varicosities by immunostaining for the vesicular ATP transporter, the solute carrier protein SLC17A9. The varicosities were similar in WT and DBA mice. Myosin Va was markedly reduced in DBA varicosities compared with the WT varicosities. Proximity ligation assay showed that myosin Va was closely associated with SLC17A9. Vesicular exoendocytosis was examined by FM1-43 staining of varicosities, which showed that exoendocytosis after KCl stimulation was impaired in DBA varicosities compared with WT varicosities. These studies show that SLC17A9 identifies ATP-containing purinergic varicosities. Myosin Va associates with SLC17A9-stained vesicles and possibly transports them to varicosity membrane for exocytosis. In myosin Va-deficient mice, purinergic inhibitory neurotransmission is impaired.