The Phtf1 gene encodes a membrane protein abundantly expressed in male germinal cells. Using a two-hybrid screening procedure we have identified FEM1B, an ortholog of the C. elegans feminization factor 1 (FEM-1), as a binding partner for PHTF1. We studied FEM1B expression in the rodent testis and found that Fem1b mRNA is present at high levels during meiosis and after, during spermiogenesis, in a similar manner to Phtf1 mRNA. Accordingly, Western blot and immunofluorescence revealed the presence of PHTF1 and FEM1B in the same cell types, and by coimmunoprecipitation we demonstrated the association between these proteins. We characterized some aspects of this interaction and showed that the ANK domain of FEM1B is necessary for the interaction with the amino extremity of PHTF1. Next, we found that FEM1B can bind several intracellular organelles and demonstrated that PHTF1 would recruit FEM1B to the endoplasmic reticulum membrane. Previous in vitro experiments had suggested that the human FEM1B was involved in apoptosis. After comparing expression profiles of FEM1B and PHTF1 with apoptotic events occurring in the normal seminiferous tubules, we suggest that neither FEM1B nor PHTF1 are directly implicated in apoptosis in this tissue.