We report the selection and characterization of influenza A/NWS-G70c and B/HK/8/73 (HG) viruses which are resistant to the potent influenza neuraminidase inhibitor, 4-guanidino-Neu5Ac2en. Viruses were selected which replicated in MDCK cells in the presence of 20 μg/ml inhibitor. The neuraminidase of resistant viruses was >200-fold more resistant to 4-guanidino-Neu5Ac2en than was the neuraminidase of the parent viruses. Although amounts of neuraminidase protein were similar in resistant and parent viruses, the enzyme activity of the resistant neuraminidase heads was reduced by >95% for the substrates used. Relative to parent viruses, the resistant viruses replicated to equal or greater titers in tissue culture and in embryonated chicken eggs. Sequence analysis revealed a single nucleotide mutation in the neuraminidase gene of each virus resulting in the change of the conserved Glu 119 (which lies in a pocket beneath the active site of the enzyme) to Gly thus eliminating an electrostatic interaction with the C-4 guanidinium moiety of the inhibitor. Mutations (Asn → Ser) at amino acids 145 and 150 were also found in the hemagglutinin gene of the B/HK/8/73 (HG) virus resistant to 4-guanidino-Neu5Ac2en. No changes were found in the hemagglutinin gene of the resistant A/NWS-G70c virus. © 1995 Academic Press. All rights reserved.