Drosophila melanogaster somatic sexual differentiation is accomplished by serial function of the products of sex-determination genes. Sex-lethal (Sxl), is one such gene. It is functionally expressed only in female flies. The sex-specific expression of this gene is regulated by alternative mRNA splicing which results in either the inclusion or exclusion of the translation stop codon containing third exon. Although previous genetic and molecular analyses suggest that functional Sxl expression is maintained by a positive feedback loop, where the female-specific Sxl product promotes the synthesis of its own female-specific mRNA, the mechanistic details of such regulation have remained unclear. We have developed a cotransfection system using Drosophila cultured (Kc) cells in which Sxl primary transcripts are expressed with or without the female specific Sxl product. Here we show that the female-specific Sxl product induces the synthesis of its own female-specific mRNA by negative control of male-specific splicing. Deletion, substitution, and binding experiments have demonstrated that multiple uridine-rich sequences in the introns around the male-specific third exon are involved in the splicing regulation of Sxl pre-mRNA.