Maintenance of synaptic transmission requires regulation of intracellular Ca(2+) in presynaptic nerve terminals; loss of this regulation at elevated temperatures may cause synaptic failure. Accordingly, we examined the thermosensitivity of presynaptic calcium regulation in Drosophila larval neuromuscular junctions, testing for effects of disrupting calcium clearance. Motor neurons were loaded with the ratiometric Ca(2+) indicator Fura-dextran to monitor calcium regulation as temperature increased. Block of the Na(+)/Ca(2+) exchanger or removal of extracellular Ca(2+) prevented the normal temperature-induced increase in resting calcium. Conversely, two treatments that interfered with Ca(2+) clearance-inactivation of the endoplasmic reticulum Ca(2+)-ATPase with thapsigargin and inhibition of the plasma membrane Ca(2+)-ATPase with high pH-significantly accelerated the temperature-induced rise in resting Ca(2+) concentration and reduced the thermotolerance of synaptic transmission. Disrupting Ca(2+)-ATPase function by interfering with energy production also facilitated the temperature-induced rise in resting [Ca(2+)] and reduced thermotolerance of synaptic transmission. Conversely, fortifying energy levels with extra intracellular ATP extended the operating temperature range of both synaptic transmission and Ca(2+) regulation. In each of these cases, Ca(2+) elevations evoked by an electrical stimulation of the nerve (evoked Ca(2+) responses) failed when resting Ca(2+) remained >e 200 nM for several minutes. Failure of synaptic function was correlated with the release of intracellular calcium stores, and we provide evidence suggesting that release from the mitochondria disrupts evoked calcium responses and synaptic transmission. Thus the thermal limit of synaptic transmission may be directly linked to the stability of ATP-dependent mechanisms that regulate intracellular ion concentrations in the nerve terminal.