A collection of 57 field isolates of the tombusvirus tomato bushy stunt virus was obtained from eggplant and tomato during 1994-1997 and was examined for the presence of defective interfering (DI) RNA species by Northern blot hybridization and RT-PCR. No DI RNA species were detected associated with any of the field TBSV isolates. However, serial passaging of two field isolates in Nicotiana clevelandii at high multiplicity of infection resulted in the rapid generation of DI-like RNA species, indicating that the absence of DI RNAs in natural populations of the virus was not due to the inability of the TBSV field isolates to generate them in a suitable host. The results indicate that DI RNAs may not play a role in modulating natural TBSV infections in the hosts examined. In 4 of 57 isolates analyzed we have detected less than full-length RNAs and we show here that they are true satellite RNAs. Two different satellite RNA species were detected, named TBSV sat RNAs B1 (822 nt) and B10 (612 nt). TBSV sat RNAs lack significant open reading frames and do not present sequence homology except in a central box that is also conserved in TBSV-Ch genomic RNA and in all the DI RNAs derived from it. TBSV sat RNA B10 attenuated the symptoms induced by the helper virus in N. clevelandii while sat RNA B1 did not modify the symptoms. This is the first report of sat RNAs associated with TBSV and the first time that sat RNAs are associated with natural tombusvirus infections.