Internal ribosome entry site (IRES) elements consist of highly structured RNA regions that determine internal initiation of translation. We have previously shown that the foot-and-mouth disease virus (FMDV) IRES contains a GNRA tetraloop spanning residues G178UAA181. Here we show that tertiary RNA interactions dependent on the GNRA motif determine the structural organization of the central domain. By using mutational analysis in combination with RNA probing, we have identified distant reciprocal interactions between the GNRA motif and the invariant region G240CACG244, termed motif A. Mutations in motif A caused a decrease in IRES activity as severe as the GUAG substitution in the GNRA motif. Substitutions in either GNRA or motif A sequences induced a common reorganization around the conserved R199AAA202 stem-loop, suggesting that the latter contributes to stabilize the GNRA-motif A interaction. This finding was also consistent with a significant increase in the efficiency of RNA-RNA interactions determined in gel shift assays using as probe the hairpin that contains the GNRA motif compared to a transcript encompassing the entire apical region of the central domain. Thus, we propose that the central domain of the FMDV IRES contains a structural conformation essential for IRES activity stabilized by a tertiary contact involving residues in the GNRA tetraloop and motif A conserved sequences.