FSHβ mRNA has a unique 3′ untranslated region (3′UTR) that is highly conserved across the species. Sequence analyses of the mouse, rat, human, bovine, and ovine 3′UTRs revealed the presence of elements implicated in mRNA instability and translational control such as AU-Rich Element (ARE) and lipoxygenase differentiation control elements. Bovine FSHβ 3′UTR down-regulated reporter expression in αT3-1 and NIH3T3 cells, but not in HEK 293 cells, suggesting the involvement of a cell-specific factor or mechanism. The presence of a 3′UTR did not influence reporter mRNA stability, but it did decrease its association with polysomes, indicating that the downregulatory effect may be exerted at the translational level. The segment spanning 601–800 bases (U4) of the bovine FSHβ 3′UTR was found to be the most effective downregulating segment, its effect being equal to that of the full-length 3′UTR. RNA electrophoretic mobility shift assay with U4 showed the presence of specific transfactors in the cytosolic preparations of bovine pituitary and the cell lines. U4 contained an ARE that appeared to be functional, because the mutated U4 ARE was ineffective in downregulating the reporter expression and inhibiting [³²P]-labeled U4-transfactor complex formation. Downregulation of reporter activity by the full-length 3′UTR and U4 could be overcome by overexpression of HuR, a protein known to stabilize ARE-containing mRNAs in NIH3T3 cells, but not in the αT3-1 cells, once again indicating that factors other than HuR may also be involved in the regulation of FSHβ in the pituitary.