Adenosine, AMP, ADP, and ATP were tested for their ability to modulate evoked quantal transmitter release at excitor-opener nerve terminals in the crayfish walking leg. Only ATP was found to have a significant effect, inhibiting release by 43%. To determine whether the effects of extracellular ATP on transmitter release were related to changes in free Ca2+ levels in the nerve terminal, net 45Ca uptake was measured in regions of the nerve near the terminal; although resting 45Ca uptake was increased by 5 mM exogenous ATP. Thus, extracellular ATP normally inhibits evoked transmitter release; this is associated with reduced stimulation-induced net Ca2+ uptake into the synaptic terminal. To explore the possibility that metabolic factors might be involved, nerve ATP levels were reduced by greater than or equal to 50% by omitting glucose from the bathing solution. Although quantal content and synaptic delay were unaffected by reduced intracellular ATP, under these conditions 5 mM exogenous ATP failed to reduce quantal release. Five millimolars of ATP did increase synaptic facilitation, however, which is consistent with a reduced ability to regulate intracellular free Ca2+. Therefore, under conditions of reduced intracellular ATP, the increased resting Ca2+ uptake produced by 5 mM ATP is not buffered completely. This leads to elevated free Ca2+ levels in the nerve terminal, increasing the amount of transmitter released following an action potential.