Identification of the 2009 H1N1 influenza A virus requires emergency use authorized (EUA) molecular reverse transcriptase-polymerase chain reaction. Laboratories lacking molecular capabilities outsource testing, which is costly and may delay result reporting. A fluorescent antibody (FA; D(3) Ultra 2009 H1N1 influenza A virus ID Kit, Diagnostic Hybrids, Athens, OH) recently received Food and Drug Administration EUA status for 2009 H1N1 virus identification. The performance of this FA reagent was evaluated in this study. Influenza A-positive nasopharyngeal specimens (seasonal H1, H3, and 2009 H1N1) were prepared for culture and FA testing and were stained using influenza A antibodies and the 2009 H1N1 reagent. Other respiratory viruses were also evaluated. The FA reagent demonstrated 100% sensitivity and specificity. Bright, apple-green fluorescence was effortlessly identified in culture-positive cells, particularly around cell membrane perimeters. Laboratory-prepared slides were preferred over bedside-prepared specimens because background fluorescence obscured identification in the latter. The new FA reagent provides an accurate, rapid, and inexpensive assay for identifying the 2009 H1N1 virus in nonmolecular diagnostic laboratories.