Over the past 35 years, developmental geneticists have made impressive progress toward an understanding of how genes specify morphology and function, particularly as they relate to the specification of each physical component of an organism. In the last 20 years, male courtship behavior in Drosophila melanogaster has emerged as a robust model system for the study of genetic specification of behavior. Courtship behavior is both complex and innate, and a single gene, fruitless (fru), is both necessary and sufficient for all aspects of the courtship ritual. Typically, loss of male-specific Fruitless protein function results in male flies that perform the courtship ritual incorrectly, slowly, or not at all. Here we describe a novel requirement for fru: we have identified a group of cells in which male Fru proteins are required to reduce the speed of courtship initiation. In addition, we have identified a gene, Trapped in endoderm 1 (Tre1), which is required in these cells for normal courtship and mating behavior. Tre1 encodes a G-protein-coupled receptor required for establishment of cell polarity and cell migration and has previously not been shown to be involved in courtship behavior. We describe the results of feminization of the Tre1-expressing neurons, as well as the effects on courtship behavior of mutation of Tre1. In addition, we show that Tre1 is expressed in a sexually dimorphic pattern in the central and peripheral nervous systems and investigate the role of the Tre1 cells in mate identification. KEYWORDS courtship; Fruitless; Drosophila; G-protein signaling; behavior O NE of the goals of neuroscience is to understand how genetic programs direct neurodevelopment and how the resulting neural pathways interact with sensory inputs to result in behavior. Drosophila melanogaster courtship behavior provides an excellent model system to study this process. Male Drosophila carry out a complex courtship ritual, the correct performance of which affects their success in mating and reproduction (Baker et al. 2001). This ritual consists of a series of stereotyped behaviors. The male first orients toward and follows the female, then taps her with his forelegs, extends one wing toward the female and vibrates it to generate a species-specific courtship song, licks her genitalia, and, finally, curls his abdomen to attempt copulation (Hall 1994). If the female is receptive (i.e., virgin or not recently mated) and the ritual is performed correctly, copulation will follow. These behavioral outputs all require sensory input and integration (Dickson 2008), and all are dependent upon expression of the male-specific isoforms of the zinc-finger transcription factor Fru (Fru M ) (Demir and Dickson 2005).The fru gene spans 130 kb of genomic DNA and generates at least 15 transcripts and six polypeptides (Demir and Dickson 2005). Most fru transcripts are common to males and females, but transcripts initiated from one of the four fru promoters (P1) are sex-specifically spliced under the control of the sexdetermining splicing ...