Previous studies have indirectly implicated the two neurotransmitters 5- HT and GABA in mediating tonic inhibition of the crayfish lateral giant (LG) escape reaction. In this study, pharmacological agents were selectively delivered to restricted portions of the abdominal CNS (where LG escape circuitry resides) to assess directly the role of these two transmitters in tonic inhibition. Both 5-HT and GABA depressed monosynaptic, electrical transmission to the LG neurons, the command neurons for LG escape, and application of either transmitter resulted in a depolarizing conductance increase in the LG neuron. The effects of 5-HT persisted in preparations in which chemical transmission was effectively abolished, implying that there are 5-HT receptors on the LG neuron itself, along with the known GABA receptors. Restricted delivery of the GABA chloride channel blocker picrotoxin to only the abdominal CNS blocked the expression of tonic inhibition there (without interfering with the rostral generation of tonic inhibition). Therefore, if 5-HT mediated tonic inhibition, the effects of 5-HT on the abdomen should also be antagonized by picrotoxin. However, this was not the case, thus suggesting that 5-HT does not mediate tonic inhibition. The most likely neurotransmitter used for tonic inhibition is GABA acting via ligand-gated chloride channels. Thus, although this form of behavioral modulation can be tonically active for very long periods, it nevertheless appears to be mediated by a classical synaptic mechanism.