A local lesion isolate of alfalfa mosaic virus (AMV-N20) from lucerne was found to encapsidate two extra RNAs in addition to the four major RNAs (RNA1, -2, -3 and -4). These were resolved by gel electrophoresis both under native conditions and after glyoxal denaturation. The RNA with an electrophoretic mobility between that of RNAs 2 and 3 was designated RNA31, that between RNAs 3 and 4 was designated RNA3s. Sucrose density gradient centrifugation analysis of AMV-N20 showed six instead of the normal four nucleoprotein components, the additional two presumably representing encapsidated RNAs 31 and 3s. RNAs 31 and 3s were both shown by Northern blot hybridization to be unrelated to host plant RNA and to contain the AMV coat protein gene sequence, which resides in RNA3. Primer extension of the RNAs 31 and 3s using a primer complementary to the 3' common terminus of all genomic AMV RNAs provided further evidence that they contained AMV sequences. RNA31 represents an addition of about 255 nucleotides, compared with RNA3, and RNA3s represents a loss of about 308 nucleotides. The coat proteins of variants encapsidating either RNA31, -3 or -3s had the same Mr indicating that the addition or deletion of the nucleotides was outside the coat protein gene. Serial mechanical passage of AMV-N20 over 5 years in four host species led both to changes in the composition of the RNA3 mixture, and to changes in symptom severity. For example, following passage in Nicotiana clevelandii, RNA3 was lost whereas passage in either N. glutinosa, Chenopodium quinoa or C. amaranticolor resulted in the loss of RNA31. No association was found between the changes in RNA3 and phenotypic changes that resulted from continuous passage for 5 years. Phenotypic changes with passage are thus presumably determined by mutations elsewhere in the virus genome.