Although much evidence suggests that calcium (Ca2+) usually triggers synaptic vesicle exocytosis and neurotransmitter release, the role of Ca2+ in vesicle endocytosis and in the delivery of fusion-competent vesicles (i.e. mobilisation and/or priming) in nerve terminals remains unclear. To address this issue, we have studied synaptic vesicle dynamics in cultured rat neurones under conditions where neurotransmitter release is triggered independently of Ca2+ using the secretagogue Ruthenium Red (RR). Using a prolonged stimulation protocol, we find that RR causes a rapid increase in neurotransmitter release followed by a gradually decrementing response. In contrast, when release is triggered by moderate membrane depolarisation caused by saline containing 18 mm K+, release is sustained. These observations suggest that when release is triggered independently of a rise in Ca2+, endocytosis or vesicle mobilisation/priming are perturbed. Using FM2-10, a fluorescent indicator of synaptic vesicle cycling, we find that neurotransmitter release triggered by RR is accompanied by both uptake and release of this dye, thereby suggesting that vesicle endocytosis is not blocked. To evaluate whether synaptic vesicle mobilisation/priming is perturbed in the absence of a rise in Ca2+, we compared the kinetics of FM2-10 loss during prolonged stimulation. While 18 mm K+ induced gradual and continuous dye loss, RR only induced substantial dye loss during the first minute of stimulation. In the presence of low concentrations of the Ca2+ ionophore ionomycin, release caused by RR was prolonged. Taken together, these results provide evidence suggesting that, although a rise in intraterminal Ca2+ is not required for endocytosis, it is essential for the continuous delivery of fusion-competent vesicles and to maintain neurotransmitter release during prolonged stimulation.