Double-stranded cDNA copies of the neuraminidase genes of influenza viruses A/Tokyo/3/67 (N2), A/tern/Australia/G70C/75 (N9), and B/Lee/40, and the hemagglutinin genes of A/Memphis/1/71 (H3) and B/Hong Kong/8/73 were cloned into a SV40 vector in which the late region was replaced by the influenza sequences. Thus the influenza genes were expressed in transfected cells under the control of the SV40 late promoter. The Tokyo/67 neuraminidase gene was modified by oligonucleotide-directed site-specific in vitro mutagenesis. Several of the amino acid residues which are conserved in all known neuraminidases and which line the sialic acid binding pocket were changed, and the mutant gene ligated back into the SV40 vector. Five mutations which have been fully characterized resulted in synthesis of a protein which had totally lost neuraminidase enzyme activity.