A presynaptic voltage-control method was used to study synaptic facilitation at the inhibitory neuromuscular synapse of the crayfish opener muscle. The expression of the F2 component of facilitation, monitored 150 ms after a conditioning stimulus, was examined by systematically changing the duration of the presynaptic test pulse. (Test pulses in all experiments were depolarized to 0 mV.) Control and facilitated inhibitory postsynaptic potentials (IPSPs) exhibited identical time courses when test pulse duration was brief (approximately 2 ms). When the duration of the test pulse was increased beyond 2 ms, the transmitter release time course shifted to an earlier point in time during facilitation. Meanwhile, the increase in total transmitter release, measured from inhibitory postsynaptic conductance (IPSG) area (total release), became less pronounced with increasing duration of the test pulse. With a 20-ms test pulse, facilitation did not cause any detectable change in total release but the half-maximal point of the facilitated IPSG shifted by 3 ms (release shift). This change in release kinetics was not associated with a decrease in minimal synaptic delay. Furthermore, the relationship between total release and presynaptic pulse duration suggested that the transmitter release activated by a 20-ms pulse can be defined as a distinct component of continuous transmitter release (early component). The facilitation process accelerated the release kinetics of the early component but did not modify its total transmitter content. To test the hypothesis that the release shift is indeed mediated by the same mechanism that increases IPSP amplitude during facilitation, we investigated the correlation between the release shift and IPSP amplitude change. The two parameters were significantly correlated when the magnitude of facilitation was changed 1) during the decay of facilitation and 2) by varying the strength of the conditioning stimulus. The experimental approach reported here provides two new physiological parameters, release shift and total release, for the analysis of synaptic facilitation.