The Drosophila fruitless (fru) gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5 -splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fru is regulated by TRA and TRA2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13-nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3 -splice site choice in the doublesex (dsx) gene. dsx has provided a useful model system to investigate the mechanisms of enhancer-dependent 3 -splice site choice. However, little is known about enhancer-dependent regulation of alternative 5 -splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant TRA/TRA2 could activate the female-specific 5 -splice site of fru. Mutational analysis demonstrated that one 13-nucleotide repeat element within the fruRE is required and sufficient to activate the regulated femalespecific splice site. As was established for dsx, the fruRE can be replaced by a short element encompassing tandem 13-nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments showed that Ser/Arg-rich proteins facilitate enhancer-dependent 5 -splice site activation. We conclude that splicing enhancers function similarly in activating regulated 5 -and 3 -splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5 -and 3 -splice sites.Alternative pre-mRNA splicing is commonly used to regulate the expression of genes and to enrich the proteomic diversity of higher eukaryotic organisms (1-4). Current estimates suggest that ϳ60% of human genes are alternatively spliced (5), sometimes leading to thousands of different mRNA isoforms from a single gene. Numerous examples describe how alternative splicing regulates gene expression in humans, but the mechanisms involved are understood in only a few cases (6 -17). In contrast, the use of genetics in Drosophila has aided in the identification of proteins that regulate alternative splicing. In particular, the genes involved in Drosophila sex determination have become prototypes for the study of positive and negative control of alternative splicing (18). The best characterized gene is the doublesex (dsx) 1 gene, in which the use of two alternative 3Ј-splice sites leads to the expression of male-or female-specific gene products that initiate gender-specific sexual differentiation. Female-specific splicing of dsx requires the production of two proteins, Transformer (TRA) and Transformer2 (TRA2), and an exonic splicing enhancer (ESE) element located within the untranslated region of the fourth exon (19 -21). Both TRA and TRA2 contain Arg/Ser-rich (RS) domains, protein interaction domains characteristic of the Ser/Arg-rich (SR) family of essential splicing factors. SR prot...