Tristetraprolin (TTP), a member of the tandem CCCH zinc finger protein family, promotes deadenylation of tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to the AU-rich elements (ARE) in their 3'-untranslated regions. The high affinity TTP-ARE binding occurs between the tandem zinc finger domain and the preferred nonamer UUAUUUAUU. By mutating a well defined core sequence of 24 bases from the tumor necrosis factor-alpha ARE, we compared the influence of four possible nonameric TTP-binding sites in the wild-type ARE with that of a single binding site in the mutated probe on the binding of TTP to the RNA and the subsequent deadenylation of the poly(A) tail. By inserting this 24-base ARE into an otherwise stable transcript, we also attempted to determine the extent of the instability conferred by the presence of one or two TTP-binding sites. These sites were created or modified by mutating the As in the UUAUUUAUU nonamer or by changing the central U in the nonamer, in both cases to C residues. The results suggest that even a single nonamer TTP-binding site can confer at least partial sensitivity to the TTP-mediated mRNA turnover on an otherwise stable mRNA, but that two binding sites make the transcript much more unstable. Even though the central U of the nonamer binding site was predicted by structural studies possibly to permit base substitution, mutation of this U to C greatly inhibited the binding of TTP to the ARE, thus reducing the ability of the TTP to promote deadenylation and instability of the mRNA.