The diverse functions of RNA, which include enzymatic activities, regulatory roles in transcription and translation, are made possible by tertiary structure. Computer algorithms can predict the secondary structure of an RNA molecule using free-energy parameters for base pairing and stacking, loops and bulges. However, with the exception of transfer RNA, little is known about the structures and thermodynamics of interactions involved in the tertiary structure of RNA. Recently, it has been proposed that a novel form of RNA folding called pseudoknotting occurs at the 3' end of certain viral RNAs from plants. A pseudoknot involves intramolecular pairing of bases in a hairpin loop with a few bases outside the stem of the loop to form an additional stem and loop region (Fig. 1). If each stem contained a full helical turn, a true knot would be formed. We present evidence from single-strand specific (S1) and double-strand specific (V1) nuclease digestion, that a short RNA oligonucleotide (19 nucleotides long) adopts a stable pseudoknotted structure. The nuclease digestion and thermodynamic properties of this oligonucleotide were compared with those of oligonucleotides which form hairpin structures containing the two possible stem regions in the pseudoknot. These results show that appropriate sequences can form pseudoknots and indicate that pseudoknots are a significant type of local tertiary structure which must be considered in the folding of complex RNA molecules.