Clinical samples from 15 amantadine-treated children were collected serially-before, during, and/or after treatment-and were studied to determine the actual prevalence, timing, and clinical implications of M2 mutational events. After viral RNA extraction and reverse-transcriptase polymerase chain reaction amplification of the viral RNA encoding the M2 protein, the products were cloned into plasmids, and their sequences were determined. Five mutations known to confer amantadine resistance in clinical samples were identified in 12 (80%) of 15 evaluable patients, and 9 patients had >1 (2-4) mutant virus. The pattern of emergence of mutant strains was clarified from the study of 6 patients with at least 4 serial samples. Although viruses with M2 mutations tended to become the dominant populations, in 2 cases, wild-type viruses became dominant after decreasing to low levels. These results suggest that resistant viruses emerge in a much higher proportion of amantadine-treated patients than has been suggested by previous studies.