Only 40 of the 359 nucleotides of Potato Spindle Tuber Viroid (PSTVd) represent the virulence-modulating (VM) region. Minor sequence variations in this domain distinguish mild from severe and even necrotic strains. Our recent hypothesis (Owens RA et al., 1996, Virology 222:144–158) that these differences result in varying degrees of bending of this part of the molecule could be tested experimentally. By in vitro transcription and partial double-strand formation, three types of model RNAs were prepared and subjected to electrophoresis in polyacrylamide gels: (1) Fragments representing the VM regions of six different PSTVd strains; (2) control fragments containing a bulge-loop as a rigid bend or an internal loop as a point of increased flexibility; and (3) dsRNAs of 36, 39, and 43 bp as length standards. Migration anomalies in gels of increasing percentage were evaluated and resulted in the following conclusions. In the absence of Mg2+, the VM regions differ only in terms of flexibility. Addition of Mg2+ induces conformational changes in these RNAs. All strains but Mild exhibit a rigid bend, and the angle of bending increases monotonically with the pathogenicity of the strain. The data are discussed in terms of a mechanism of pathogenicity, that protein-binding to the VM region is the primary pathogenic event.