Our recent study showed a possibility that newly developed A2 type botulinum toxin (A2NTX) inhibits both spontaneous and evoked transmitter release from inhibitory (glycinergic or GABAergic) and excitatory (glutamatergic) nerve terminals using rat spinal sacral dorsal commissural nucleus neurons. In the present study, to determine the modulatory effect of A2NTX on glycinergic and glutamatergic release probabilities, we tested the effects of A2NTX on a single inhibitory or excitatory nerve ending adherent to a dissociated neuron that was activated by paired-pulse stimuli by using the focal electrical stimulation technique. The results of the present paired-pulse experiments showed clearly that A2NTX enhanced paired-pulse facilitation of evoked glycinergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents and increased the failure rate (Rf) of the first postsynaptic currents (P1) and both the responses. These effects of A2NTX on the amplitude and Rf of the P1 and the second postsynaptic currents (P2) and paired-pulse ratio were rescued by application of 4-aminophthalimide. In summary, the present results showed that A2NTX acts purely presynaptically and inhibits the release machinery of transmitters such as glycine and glutamate, and the transmitter release machinery became less sensitive to intracellular free-Ca2+ in A2NTX poisoned nerve terminals.