The effects of temperature and type of host on the evolution of variants in replicating populations of tobacco mosaic virus (TMV) originating from an in vitro RNA transcript of a cDNA clone were studied. The phenotypic markers measured were temperature-sensitive (ts) replication and induction of necrotic local lesions (nl) on Nicotiana sylvestris. The proportions of ts variants were about the same under all conditions tested, but changes in temperature or host had strong effects on proportions of nl variants. During replication at elevated temperatures in tobacco or tomato, percentages of nl variants were reduced rapidly to about one tenth their initial values. In contrast, when virus replicated at 25 degrees in tobacco, little change in the average proportion of nl variants was observed, and in tomato, only a slight average reduction occurred. Virus replicating at 25 degrees in Solanum nigrum or Petunia hybrida suffered effects similar to those observed in tobacco or tomato, respectively. However, at 25 degrees in Physalis floridana, there was strong selection against nl variants, which eventually reduced them to essentially undetectable levels. In all hosts tested at 25 degrees, surprisingly large, apparently random changes in the proportion of nl variants occurred in individual plants. These experiments showed that virus populations can evolve rapidly on a time scale of days, and that an element of randomness is an important component in the initiation of change.