Full-length complementary DNAs (cDNA) of a mild (KF5) and a severe (S-PSTVd) isolate of potato spindle tuber viroid (PSTVd) were constructed. DNA sequencing of four KF5 cDNA clones (M3, M4, M5, and M7) revealed that KF5 is comprised of 360 nucleotides. By comparison, all three cDNA clones (S2, S9, and S10) of S-PSTVd possess 359 nucleotides. Sequence microheterogeneity was observed among the KF5 cDNA clones. Clone M5 differs from mild PSTVd isolate KF6 by a U-to-A transversion at position 303 followed by an A addition at the lower half of the "virulence-modulating" (VM) region. These changes modified the PSTVd consensus sequence of the VM region from 5' UCUAUCU 3' to 5' UCAAAUCU 3'. Additionally, clones M4 and M7 have a G-to-A transition at position 65 of the pathogenic domain, and M3 has a G-to-A transition at position 133 of the variable domain. The sequence of the three cDNA clones of S-PSTVd was identical to that of PSTVd isolate 440-1. An improved computer program was used to predict the secondary structure of the above two sequence variants as well as that of other PSTVd variants of which the structure has been reported previously. The data provides support for the hypothesis that increasing thermodynamic instability of the VM region is correlated with increasing virulence of the respective naturally occurring PSTVd isolate.