The locus ovarian tumor (otu) is involved in several aspects of oogenesis in Drosophila melanogaster. The possible role of otu in the determination of the sexual identity of germ cells has not been extensively explored. Some otu alleles produce a phenotype known as ovarian tumors: ovarioles are filled with numerous poorly differentiated germ cells. We show that these mutant germ cells have a morphology similar to primary spermatocytes and that they express male germ line-specific reporter genes. This indicates that they are engaged along the male pathway of germ line differentiation. Consistent with this conclusion, we found that the splicing of Sex-lethal (Sxl) pre-mRNAs occurs in the male-specific mode in otu-transformed germ cells. The position of the otu locus in the regulatory cascade of germ line sex determination has been studied by using mutations that constitutively express the feminizing activity of the Sxl gene. The sexual transformation of the germ cells observed with several combinations of otu alleles can be reversed by constitutive expression of Sxl. This shows that otu acts upstream of Sxl in the process of germ line sex determination. Other phenotypes of otu mutations were not rescued by constitutive expression of Sxl, suggesting that several functions of otu are likely to be independent of sex determination. Finally, we show that the gene dosage of otu modifies the phenotype of ovaries heterozygous for the dominant alleles of ovo, another gene involved in germ line sex determination. One dose of otu+ enhances the ovoD ovarian phenotypes, while three doses partially suppress these phenotypes. Synergistic interaction between ovoD1 and otu alleles leads to the occasional transformation of chromosomally female germ cells into early spermatocytes. These interactions are similar to those observed between ovoD and one allele of the sans fille (snf) locus. Altogether, our results imply that the otu locus acts, along with ovo, snf, and Sxl, in a pathway (or parallel pathways) required for proper sex determination of the female germ line.