The tetracycline aptamer is an in vitro selected RNA that binds to the antibiotic with the highest known affinity of an artificial RNA for a small molecule (Kd ∼0.8 nM). It is one of few aptamers known to be capable of modulating gene expression in vivo. The 2.2 Å resolution cocrystal structure of the aptamer reveals a pseudoknot-like fold formed by tertiary interactions between an 11 nucleotide loop and the minor groove of an irregular helix. Tetracycline binds within this interface as a magnesium ion chelate. The structure, together with previous biochemical and biophysical data, indicates that the aptamer undergoes localized folding concomitant with tetracycline binding. The three-helix junction, h-shaped architecture of this artificial RNA is more complex than those of most aptamers and is reminiscent of the structures of some natural riboswitches.