This report describes the clinical evaluation of a novel fluorescent immunoassay (FIA), Sofia Influenza A+B FIA (Quidel, San Diego, CA), for the rapid detection and differentiation of influenza A and B viruses. A total of 2,047 subjects provided nasal swabs and nasopharyngeal swabs or aspirates. The overall sensitivity and specificity for influenza A virus vs virus culture were 94% and 95%, respectively, and for influenza B virus were 89% and 96%, respectively. Fourteen hundred and sixty-one specimens were available for testing with reverse transcriptase-polymerase chain reaction (RT-PCR). The sensitivity of the Sofia Influenza A+B FIA for detecting influenza A and B viruses compared with the RT-PCR method was 78% and 86%, respectively. A high percentage of the positive specimens had low cycle threshold values, and almost all of these were positive with the Sofia test. This high level of sensitivity demonstrates that the Sofia influenza A+B FIA could improve the usefulness of rapid influenza virus testing.