Follicle stimulating hormone (FSH) is essential for normal gonadal function in mammals. Expression of its beta subunit (FSHB) controls overall production/secretion of FSH and is induced by activin. Studies with ovine FSHB promoter/reporter constructs in LT2 gonadotropes show that induction by activin requires a putative Smad binding element (SBE) in the ovine FSHB promoter (-162AGAC-159). Similar studies reported here show that another site, juxtaposed to the SBE was also required for 81% of activin induction in LT2 cells. This site was similar to several that bind proteins known to partner with Smads. When this site (-171ACTgcgtTT-163) was mutated by changing the nucleotides shown in lower case letters, the resulting ovine-derived construct (mut-oFSHBLuc) was expressed poorly as a transgene in primary mouse gonadotropes (< 0.001x compared to ovine wild type transgenes). This decrease in expression demonstrated the importance of this site for activin induction and, perhaps, basal expression although studies with LT2 cells did not suggest this latter possibility. Expression of mut-oFSHBLuc in male mouse gonadotropes in vivo was 644x expression in all but one non-gonadotrope tissue tested indicating that mut-oFSHBLuc retained significant gonadotrope-specific expression. A rise in FSHB expression occurs during estrus in mice and is faithfully reproduced with wild type ovine FSHBLuc transgenes, but not with mut-oFSHBLuc, indicating that the mutated site is needed for this secondary FSH surge. These data suggest that activin gathers Smads and Smad associated proteins at the -171/-159 promoter region to regulate expression of the ovine FSHB subunit and overall FSH production.