Diagnosis of respiratory syncytial virus (RSV) during acute infection in adults is difficult because of the poor sensitivity of viral culture and antigen detection. A recently developed single-tube nested reverse transcription-PCR (RT-PCR) was compared to viral culture and serology by enzyme immunoassay for the diagnosis of RSV in adults with respiratory illness. Nasal swab samples were collected during respiratory illnesses from five groups of subjects: healthy young adults, healthy elderly adults, adults with chronic heart and lung disease, nursing home residents, and adults admitted to the hospital during the winter with cardiopulmonary illnesses. Of 1,112 samples for which all three tests were available, 117 were positive by at least one method and 995 were negative by all methods. One hundred ten were considered true positives because culture and/or serology was positive. Of these, 80 (73%) were PCR positive compared to 43 (39%) that were culture positive. Seven PCR results were considered false positives due to negative culture and serology. The overall RT-PCR sensitivity was 73%, and specificity was 99%. These data indicate that RT-PCR is an excellent method for the diagnosis of acute RSV infection in adults.