The sialidase (neuraminidase) inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-guanidino-Neu5Ac2en) has been examined for the ability to inhibit the growth of a wide range of influenza A and B viruses in vitro in comparison with amantadine, rimantadine, and ribavirin. 4-Guanidino-Neu5Ac2en inhibited plaque formation by laboratory-passaged strains of influenza A and B viruses, with 50% inhibitory concentrations ranging from 0.005 to 0.014 microM. A wider range of values (0.02 to 16 microM) was obtained with more recent clinical isolates, but in all cases 4-guanidino-Neu5Ac2en inhibited influenza A and B virus replication at lower concentrations than amantadine, rimantadine, or ribavirin. Inhibition by 4-guanidino-Neu5Ac2en was not obviously affected by the passage history of the viruses or by resistance to amantadine or rimantadine. 4-Guanidino-Neu5Ac2en was a very potent inhibitor of the sialidases of all the influenza viruses examined, with 50% inhibitory concentrations ranging from 0.00064 to 0.0079 microM. No cytotoxicity was observed with 4-guanidino-Neu5Ac2en at up to 10 mM. 4-Guanidino-Neu5Ac2en therefore represents a new potent and selective inhibitor of influenza A and B virus sialidase activity and replication in vitro.