Robust innate behaviours are attractive systems for genetically dissecting how environmental cues are perceived and integrated to generate complex behaviours. During courtship, Drosophila males engage in a series of innate, stereotyped behaviours that are coordinated by specific sensory cues. However, little is known about the specific neural substrates mediating this complex behavioural programme. Genetic, developmental and behavioural studies have shown that the fruitless (fru) gene encodes a set of male-specific transcription factors (FruM) that act to establish the potential for courtship in Drosophila. FruM proteins are expressed in approximately 2% of central nervous system neurons, at least one subset of which coordinates the component behaviours of courtship. Here we have inserted the yeast GAL4 gene into the fru locus by homologous recombination and show that (1) FruM is expressed in subsets of all peripheral sensory systems previously implicated in courtship, (2) inhibition of FruM function in olfactory system components reduces olfactory-dependent changes in courtship behaviour, (3) transient inactivation of all FruM-expressing neurons abolishes courtship behaviour, with no other gross changes in general behaviour, and (4) 'masculinization' of FruM-expressing neurons in females is largely sufficient to confer male courtship behaviour. Together, these data demonstrate that FruM proteins specify the neural substrates of male courtship.