The effect of increasing [Ca2+]o on the positioning of synaptic vesicles relative to the active site in resting coxal neuromuscular junctions of Drosophila was investigated. In normal saline (1.8 mM Ca2+) only a very small percentage of sites possess a vesicle docked under the dense body plate close to the plasma membrane in a readily releasable position. However, after exposure to elevated Ca2+ salines (3.6, 9, 18 mM), an increase in the number of active sites possessing docked vesicles was observed. Also, an increase in the average number of docked vesicles/site was seen. Intracellular recordings from coxal muscle fibers in normal saline and elevated Ca2+ salines were made, and it was observed that exposure to elevated Ca2+ saline caused an increase in miniature excitatory junction potential (mejp) frequency and in multiquantal and clustered mejps. Thus, when the number of active sites possessing docked vesicles increases, the frequency of spontaneous release also increases. Furthermore, when the number of docked vesicles/site increases, the number of multiquantal mejps increases. The data suggest that Ca2+ may be involved in vesicle translocation to the active site, and that the concentration of Ca2+ in the terminal may regulate the number of active sites that possess readily releasable vesicles. The effects of increasing the number of docked vesicles on spontaneous release characteristics are discussed.