Expression of the putative replicase of potato leafroll virus (PLRV) is regulated by -1 ribosomal frameshifting in which a primary viral transcript has two overlapping open reading frames (ORFs). A region of 39 nt at the junction of the two ORFs is essential for frameshifting to occur. It has been shown to harbor two signals, one active on the level of the primary structure, termed the slippery sequence, and one component that forms a secondary or tertiary level structure, described as either a pseudoknot or a stem-loop motif. We have performed extensive site-directed mutagenesis of the frameshifting region and analyzed individual mutants for their ability to promote -1 frameshifting in vitro. Detailed comparison of our results with analogous mutants in the frameshifting region of the evolutionarily related beet western yellow virus, for which a crystal structure is available, unequivocally argues for the pseudoknot to be the structural motif necessary for the frameshifting function in PLRV transcripts. Mutations in PLRV that affect putative pseudoknot-specific tertiary-base interactions drastically affect frameshifting activity. In addition, a specific deletion mutant was identified that displayed PLRV wild-type frameshifting activity with only 22 nt available for pseudoknot formation.