Insertional mutagenesis using P-element vectors yielded several independent mutations that cause male homosexuality in Drosophila melanogaster. Subsequent analyses revealed that all of these insertions were located at the same chromosomal division, 91B, where one of the inversion breakpoints responsible for the bisexual phenotype of the fruitless (fru) mutant has been mapped. In addition to the altered sexual orientation, the fru mutants displayed a range of defects in the formation of a male-specific muscle, the muscle of Lawrence. Since the male-specific formation of this muscle was dependent solely on the sex of the innervating nerve and not on the sex of the muscle itself, the primary site of action of the fru gene should be in the neural cells. satori, one of the P-insertion alleles of fru which we isolated, carried the lacZ gene of E. coli as a reporter, and beta-galactosidase expression was found in a subset of brain cells including those in the antennal lobe in the satori mutant. Targeted expression of a sex determination gene, transformer (tra), was used to produce chromosomally male flies with certain feminized glomeruli in the antennal lobe. Such sexually mosaic flies courted not only females but also males when the DM2, DA3 and DA4 glomeruli were feminized, indicating that these substructures in the antennal lobe may be involved in the determination of the sexual orientation of flies. Molecular cloning and analyses of the genomic and complementary DNAs indicated that transcription of the fru locus yields several different transcripts, one of which encodes a putative transcription regulator with a BTB domain and two zinc finger motifs. In the 5' non-coding region, three putative Transformer binding sites were identified. It appears plausible therefore that the fru gene is one of the elements in the sex determination cascade that controls sexual fates of certain neuronal cells. Improper sex determination in these neural cells may lead to altered sexual orientation and malformation of the male-specific muscle. Some implications of the results of our study on sexual orientation in other organisms will be discussed based on the Drosophila research.