The innate immune system mounts the first host response to pathogens. Because alpha-defensins, which are cationic antimicrobial peptides of polymorphonuclear neutrophils and other leukocytes, are important effectors of the innate immune system, we studied the antiviral activity of human alpha-defensin-1 (also known as "human neutrophil peptide-1" [HNP-1]) against influenza virus in vitro. Treatment of cell cultures with HNP-1 soon after infection resulted in marked inhibition of influenza virus replication and viral protein synthesis. This effect was not due to cytotoxicity or to a direct effect on the virus. Treatment of cells with HNP-1 followed by its removal before infection also inhibited viral replication, suggesting that the inhibition was due to the modulation of cellular pathways. HNP-1 treatment inhibited protein kinase C (PKC) activation in infected cells, suggesting the involvement of the PKC pathway. Our data expand the previously known activity of alpha -defensins against influenza virus. Characterizing the mechanism of action of alpha -defensins may lead to the identification of new strategies for prevention and therapy.