More than half of the identified ascending interneurons originating in the terminal abdominal ganglion of the crayfish received inhibitory sensory inputs from hair afferents innervating the tailfan on the side contralateral to their main branches. Biochemical aspects of this transverse lateral inhibition of ascending interneurons were examined by the use of neurophysiological and pharmacological techniques. Local application of gamma-aminobutyric acid (GABA) and its agonist muscimol into the neuropil induced membrane hyperpolarization of identified ascending interneurons with an increase in membrane conductance. Because the reversal potential of inhibitory postsynaptic potential (IPSPs) in ascending interneurons elicited by the sensory stimulation and GABA injection was similar, and the sensory-stimulated IPSPs of the interneurons were blocked by GABA and muscimol application, this study strongly suggests a GABAergic nature for transverse lateral inhibition of ascending interneurons. According to the response to the GABAA antagonists bicuculline and picrotoxin, ascending interneurons were classified into two types, picrotoxin-sensitive and picrotoxin-insensitive interneurons. Identified ascending interneurons VE-1 and RO-4 showed a pharmacological profile similar to that of the classical GABAA receptor of the vertebrates. Bath application of both bicuculline and picrotoxin reversibly reduced the amplitudes of IPSPs. The other identified ascending interneurons CA-1, RO-1, and RO-2 were not affected significantly by the bath application of GABAA and GABAB antagonists, although bath application of low-chloride saline reversed the sensory-stimulated IPSPs. IPSPs of the picrotoxin-sensitive interneurons had a rather faster time course and shorter duration in comparison with those of the picrotoxin-insensitive interneurons.