Utilization of tightly linked ovo-A vs. ovo-B germline promoters results in the expression of OVO-A and OVO-B, C(2)H(2) transcription factors with different N -termini, and different effects on target gene transcription and on female germline development. We show that two sex-determination signals, the X chromosome number within the germ cells and a female soma, differentially regulate ovo-B and ovo-A. We have previously shown that OVO regulates ovarian tumor transcription by binding the transcription start site. We have explored the regulation of the ovo-B promoter using an extensive series of transgenic reporter gene constructs to delimit cis-regulatory sequences as assayed in wild-type and sex-transformed flies and flies with altered ovo dose. Minimum regulated expression of ovo-B requires a short region flanking the transcription start site, suggesting that the ovo-B core promoter bears regulatory information in addition to a "basal" activity. In support of this idea, the core promoter region binds distinct factors in ovary and testis extracts, but not in soma extracts, suggesting that regulatory complexes form at the start site. This idea is further supported by the evolutionarily conserved organization of OVO binding sites at or near the start sites of ovo loci in other flies.