The genetically programmed courtship song of male Drosophila melanogaster has a rhythmic repetitive structure of sound pulses that is a critical component of mate recognition. This study investigates the neuronal basis of courtship song production by using the GAL4 enhancer trap paradigm to drive the expression of UASG-tetanus toxin (TNT) and UASG-transformer (tra) in subsets of cells. Of the 727 enhancer trap lines screened for courtship song defects in this way, 38 produced a phenotype, and these were used to drive the expression of Green Fluorescent Protein (GFP), thereby implicating the corresponding neuronal structures. Twelve of 13 lines which gave vigorous courtship but did not produce song, showed GFP expression in the mushroom bodies (MBs) and the giant descending neuronal tract and/or the larval interhemispheric junction (IJ). Furthermore, the relevant DNA in which the enhancer trap was inserted was isolated and sequenced in 27 of the lines, and a number of candidate genes that have been implicated in Ras and hedgehog signalling were identified. Other candidate genes that could plausibly affect courtship song were involved in mesothoracic or axonal development.