Velvet tobacco mottle virus (VTMoV) isolated from Nicotiana velutina growing wild in arid Central Australia was transmitted by inoculation to a limited number of plant species of which N. clevelandii was the most convenient experimental host. The virus was also transmitted from field-grown plants toN. velutina and N. clevelandii by the mirid Cyropeltis nicotianae. VTMoV preparations purified by clarification with organic solvents and differential centrifugation contained polyhedral particles about 30 nm in diameter sedimenting as a single component at about 115 S. The particles were shown to be located in the nucleus, cytoplasm, and vacuoles of infected plant cells. Virus dissociated in the presence of mercaptoethanol and sodium dodecyl sulfate (SDS) separated into one major and two minor polypeptides with estimated molecular weights of 33,000, 36,000 and 31,000, respectively. Single-stranded RNA isolated from VTMoV by extraction with phenol was separated into five components with apparent molecular weights of 1.5 x 10(6), 0.63 x 10(6), 0.25 x 10(6), 0.16 x 10(6), and 0.12 x 10(6) referred to as RNAs 1, 1a, 1b, 2, and 3, respectively. It appears that RNAs 1a and 1b are breakdown products of RNA 1, as shown elsewhere, and electron microscopic examination of the other species showed that whereas RNAs 1 and 3 are linear molecules, RNA 2 is circular. The similarity of RNAs 2 and 3 to the RNA of viroids is discussed. VTMoV has been compared with several RNA plant viruses with small polyhedral particles. Only solanum nodiflorum mottle virus appears to share some of its unique features and the two have been shown to be antigenically related.