The female-sterile ovarian tumor gene, otu, is located in cytological region 7F1 on the Drosophila melanogaster chromosome map. We have mapped the gene at the molecular level by using four dysgenic alleles and two revertant derivatives of these alleles as well as an ethyl methanesulfonate-induced allele. The insertional (dysgenic) changes were all associated with one restriction fragment, and its size was restored after phenotypic reversion. One ethyl methanesulfonate-induced allele had a deletion in the restriction fragment adjacent (distal) to the fragment altered in the insertional alleles. These two restriction fragments were immediately adjacent to the s38 chorion gene. Associated with the two altered restriction fragments were two RNA species, an abundant 3.2-kilobase (kb) poly(A)+ RNA and a minor 4.0-kb RNA. Several other less-abundant RNA species were detectable with more-sensitive single-stranded RNA probes. The otu gene was transcribed proximal to distal relative to the centromere; this was opposite to the direction of transcription of the adjacent s38 gene. During development, the 3.2-kb RNA was absent in larvae, first appeared in the pupal stages, and persisted in adult females, in which it was most prevalent in the ovaries. The DNA that hybridized to the 3.2-kb ovarian RNA hybridized to four different RNAs found in the testes but not in the rest of the adult male. These testis-enriched RNAs were transcribed from the same strand of DNA as the ovarian transcripts.