The relationship between external Ca and twin pulse facilitation of transmitter release has been investigated in neuromuscular junctions of Drosophila larvae using external electrodes to record excitatory junctional currents (ejcs). The degree of synaptic facilitation decreases in inverse relation to [Ca2+]0, even at levels of transmitter release that do not cause depletion of quanta. This reverse-dependency of facilitation to external calcium is abolished by the slow mutation which suppresses the transient Ca-dependent K current (ICF). A likely explanation for these observations is that the activation of ICF by residual Ca shortens the action potential duration resulting in a reduced release of transmitter to the test stimulus. This represents a negative feed-back mechanism of synaptic function which opposes synaptic facilitation.