In the first step toward identifying the neurotransmitter released from spiking interneurons of both local and intersegmental groups in the crayfish terminal abdominal ganglion, the authors examined whether spiking local interneurons and ascending intersegmental interneurons contain the transmitter โฅ-aminobutyric acid (GABA). In this paper, 17 identified ascending interneurons and three spiking local interneurons were stained by intracellular injection of Lucifer yellow and subsequently treated for immunocytochemical staining against GABA. Double-labeling experiments revealed that six identified ascending interneurons are GABAergic, but no spiking local interneurons show GABA-like immunoreactivity. Four ascending interneurons with GABAlike immunoreactivity (reciprocal closing ascending neuron 5 [RC-5], reciprocal opening ascending neuron 6 [RO-6], variable-effect ascending interneuron 1 [VE-1], and no-effect ascending interneuron 4[NE-4]) had cell bodies that formed a cluster on the ventral surface of the rostral edge of the ganglion, whereas two GABAergic interneurons (coinhibiting ascending interneuron 2 [CI-2] and NE-2) had cell bodies in a caudal region around the cell body of the seventh flexor inhibitor (FI) motor neuron. Another four rostral interneurons (RC-2, RC-3, RC-4, and NE-3) and seven caudal interneurons (CI-3, RC-7, RO-1, RO-2, RO-3, RO-4, and NE-1) had no GABA-like immunoreactivity. Because VE-1 is known to make direct inhibitory connections with other ascending interneurons, whereas RC-3 and RO-1 are known to make direct excitatory connections, the immunocytochemical results from this study are consistent with previous physiological studies. Although many spiking local interneurons (including spiking local interneuron 1 of the anterior group [sp-ant1]) made direct inhibitory connections with nonspiking local interneurons, three spiking local interneurons (sp-ant1, spiking local interneuron 6 of the medial group [sp-med6], and spiking interneuron 5 of the posterior group [sp-post]) do not show GABA-like immunoreactivity. These results suggest that the inhibitory transmitter released from spiking local interneurons is not GABA but that another substance mediates the inhibitory action of these interneurons.