Presynaptic inhibition is produced by increasing Cl(-) conductance, resulting in an action potential of a smaller amplitude at the excitatory axon terminals. This, in turn, reduces Ca(2+) entry to produce a smaller release. For this mechanism to operate, the "inhibitory" effect of shunting should last during the arrival of the "excitatory" action potential to its terminals, and to achieve that, the inhibitory action potential should precede the excitatory action potential. Using the crayfish neuromuscular preparation which is innervated by one excitatory axon and one inhibitory axon, we found, at 12 degrees C, prominent presynaptic inhibition when the inhibitory action potential followed the excitatory action potential by 1, and even 2, ms. The presynaptic excitatory action potential and the excitatory nerve terminal current (ENTC) were not altered, and Ca(2+) imaging at single release boutons showed that this "late" presynaptic inhibition did not result from a reduction in Ca(2+) entry. Since 50 microM picrotoxin blocked this late component of presynaptic inhibition, we suggest that gamma-aminobutyric acid-A (GABA(A)) receptors reduce transmitter release also by a mechanism other than affecting Ca(2+) entry.