Female adnexal tumors of probable wolffian origin (FATWOs) are rare. They can closely mimic endometrioid adenocarcinomas with a prominent spindle cell component and Sertoli cell tumors (SCTs). To further define their immunohistochemical profile and origin, we investigated the expression of PAX-8, PAX-2, and GATA binding protein 3 (GATA-3) (wolffian markers) and of steroidogenic factor-1 (SF-1) (sex-cord stromal marker) in FATWOs. We also studied the expression of PAX-8 and PAX-2 in endometrioid adenocarcinomas; of SF-1 in Sertoli-Leydig cell and SCTs; and of PAX-8, PAX-2, GATA-3, and SF-1 in rete ovarii-a proposed site of origin for FATWOs. A database search yielded 8 FATWOs, 18 ovarian/tubal/paraovarian endometrioid adenocarcinomas, and 8 ovarian Sertoli-Leydig cell and SCTs. Eleven cases with rete ovarii sections were included. Of the FATWOs studied, all were negative for PAX-8, PAX-2, GATA-3, and SF-1. Of the endometrioid adenocarcinomas studied, PAX-8 was positive in all and PAX-2 was positive in 57%. Of the Sertoli-Leydig cell and SCTs, all were positive for SF-1 except one. The rete ovarii were positive for PAX-8, weakly positive for SF-1, and negative for PAX-2 and GATA-3. Our study suggests that PAX-8 and SF-1 can be helpful in the distinction between FATWOs and endometrioid adenocarcinomas and SCTs, respectively. Our results do not support a Mullerian or sex-cord stromal or rete ovarii origin for FATWOs. It is curious, however, that FATWOs do not express wolffian markers-it is possibly related to their origin from a distinctive portion of the wolffian duct.