Sixty-four single plaque subclones of a small-plaque-forming mutant of encephalomyocarditis virus, EMC/r+, were isolated and titrated. In addition to EMC/r+ virus, some contained large-plaque-forming virus, EMC/r. The selective conditions which prevailed during the growth and isolation of the subclones were analysed in detail. All the evidence suggests that only negligible differential selection favouring either the parental (EMC/r+) or the newly arisen plaque type (EMC/r) could have taken place. The changing selective conditions which operate during the growth of EMC/r mutants arising during the plaque assay of EMC/r+, and which slow down their emergence, are discussed. The changes in plaque morphology were shown to be genetically determined. For estimation of the mutation rate the subclones were placed a priori into one of three groups according to their total virus (infectivity) content. The mutation rate from r+ → r was calculated by four methods from each of the 3 groups and found to be 1–2 × 10-4 per particle per duplication.