The roles of the transcription factor Engrailed (En), and its paralogue Invected (Inv), in adult Drosophila Johnston's Organ sensory neurons are unknown. We used en-GAL4 driven CD8-GFP and antibody staining to characterize these neurons in the pedicel (second antennal segment). The majority of En and Inv-expressing Johnston's Organ neurons (En-JONs) are located in the ventral part of the posterior group of JONs, with only a few in the medial group. Anatomical classification of En-JON axon projections shows they are mainly type A and E, with a few type B. Extracellular recording of sound-evoked potentials (SEPs) from the antennal nerve was used along with Kir2.1 silencing to assess the contribution that En-JONs make to the auditory response to pure-tone sound stimuli. Silencing En-JONs reduces the SEP amplitude at the onset of the stimulus by about half at 100, 200 and 400 Hz, and also reduces the steady-state response to 200 Hz. En-JONs respond to 82 dB and 92 dB sounds but not 98 dB. Despite their asymmetrical distribution in the Johnston's Organ they respond equally strongly to both directions of movement of the arista. This implies that individual neurons are excited in both directions, a conclusion supported by reanalysis of the morphology of the pedicel-funicular joint. Other methods of silencing the JONs were also used: RNAi against the voltage-gated Na⁺ channel encoded by the para gene, expression of attenuated diphtheria toxin, and expression of a modified influenza toxin M2(H37A). Only the latter was found to be more effective than Kir2.1. Three additional JON subsets were characterized using Flylight GAL4 lines. inv-GAL4 88B12 and Gycβ100B-GAL4 12G03 express in different subsets of A group neurons and CG12484-GAL4 91G04 is expressed in B neurons. All three contribute to the auditory response to 200 Hz tones.