We surveyed the incidence of amantadine-resistant influenza A viruses both at sentinel surveillance sites and at nursing homes, and verified their types of change by partial nucleotide sequence analysis of the M2 protein. Fifty-five influenza A viruses from 27 sentinel surveillance sites during six influenza seasons from 1993 to 1999, and 26 influenza A viruses from 5 nursing homes from 1996 to 1999 were examined for susceptibility to the drug by virus titration in the presence or absence of amantadine. While amantadine-resistant viruses were not found in sentinel surveillance sites, a high frequency of resistance (8/26, 30.8%) in nursing homes was observed. Resistant viruses can occur quickly and be transmitted when used in an outbreak situation at nursing homes, where amantadine is used either for neurologic indications or for influenza treatment. Eight resistant viruses had a single amino acid change of the M2 protein at residue 30 or 31. In vitro, all 11 sensitive viruses turned resistant after 3 or 5 passages in the presence of 2 microg/ml amantadine, and they showed an amino acid change at residue 27, 30, or 31. The predominant amino acid substitution in the M2 protein of resistant viruses is Ser-31-Asp (a change at 31, serine to asparagine). The results indicate that a monitoring system for amantadine-resistant influenza viruses should be established without delay in Japan.