Previously we constructed an infectious hop stunt viroid (HSV) cDNA clone, PHS-P2P, which carries two copies of full-length HSV cDNA tandemly and generates HSV RNA when it is inoculated into cucumber plants. The in vitro transcript of the cDNA clone was also infectious. To investigate the essential regions for infectivity of HSV, we introduced a short deletion or insertion into the HSV sequence of pHS-P2P using restriction sites (XhoI site for pHI-X1, PvuI site for pHI-P1, and BamHI site for pHI-B1) and assayed the infectivities of these mutagenized clones. None of these mutagenized clones and their transcripts were infectious under the conditions used. Simultaneous inoculation of two or three of non-infectious mutagenized clones or the transcripts from them did not restore the infectivity.