Avian influenza A virus subtype H5N1 transmission to domestic cats and other felids has created concern because highly pathogenic avian H5N1 virus can cause fatal infections in humans. Experimental infections have demonstrated transmission of influenza viruses in cats. In this study, an epidemiologic survey of feral cats was conducted to determine their exposure to influenza A virus. Feral cat serum samples and oropharyngeal and rectal swabs were collected from November 2008 through July 2010 in Alachua County, Florida and were tested for evidence of influenza A virus infection. No virus was isolated from any of 927 cats examined using MDCK cell or embryonated chicken egg culture methods, nor was viral RNA detected by RT-PCR in 200 samples tested. However, 0.43% of cats tested antibody positive for influenza A by commercial ELISA. These results suggest that feral cats in this region of Florida are at minimal risk for influenza A virus infection.