During courtship, male Drosophila melanogaster sing a multipart courtship song to female flies. This song is of particular interest because (1) it is species specific and varies widely within the genus, (2) it is a gating stimulus for females, who are sensitive detectors of conspecific song, and (3) it is the only sexual signal that is under both neural and genetic control. This song is perceived via mechanosensory neurons in the antennal Johnston's organ, which innervate the antennal mechanosensory and motor center (AMMC) of the brain. However, AMMC outputs that are responsible for detection and discrimination of conspecific courtship song remain unknown. Using a large-scale anatomical screen of AMMC interneurons, we identify seven projection neurons (aPNs) and five local interneurons (aLNs) that outline a complex architecture for the ascending mechanosensory pathway. Neuronal inactivation and hyperactivation during behavior reveal that only two classes of interneurons are necessary for song responses--the projection neuron aPN1 and GABAergic interneuron aLN(al). These neurons are necessary in both male and female flies. Physiological recordings in aPN1 reveal the integration of courtship song as a function of pulse rate and outline an intracellular transfer function that likely facilitates the response to conspecific song. These results reveal a critical pathway for courtship hearing in male and female flies, in which both aLN(al) and aPN1 mediate the detection of conspecific song. The pathways arising from these neurons likely serve as a critical neural substrate for behavioral reproductive isolation in D. melanogaster.