Influenza A virus remains an important human and zoonotic pathogen, causing both seasonal outbreaks of illness and rare pandemics. The disease is currently managed through vaccination and neuraminidase-inhibiting pharmaceuticals. In recent years, our understanding of the role that neuraminidase (NA) plays in viral infection has broadened to include assisting virus through the sialic acid-rich mucus overlying epithelial cells of the respiratory system. The sialidase activity of influenza virus against human-like (alpha 2,6-linked) and avian-like (alpha 2,3-linked) sialic acid substrates varies between different virus strains. All are thought to exhibit cleavage of a-2,3-linked sialic acids but additional activity against a-2,6-linked sialic acids is thought to increase as the virus evolves within its human host. What influence, if any, that influenza NA specificity has in host cell infection is not completely understood. In this study, reverse engineered viruses with substitution of and/or mutations in the neuraminidase gene have been produced to examine different neuraminidase specificities and activities on an otherwise constant genetic background. We then evaluated the enzymatic activity of NA variants against human and avian-like sialic acid substrates and viral replication kinetics in vitro and in vivo. Substitution of NA in A/Udorn/307/72 (H3N2) with NA from A/Bangkok/1/79 (H3N2) led to an overall decrease in NA activity but no change in NA specificity. This swapping of NA also resulted in decreased growth in embryonated chicken eggs and BALB/c mice (both having a-2,3-linked sialic acids) but surprisingly not in MDCK (dual receptors). Viruses with single point mutations in the amino acid sequences of Udorn and Bangkok NA active sites have also been produced and show changes in NA activity or specificity or resulted in a non-functional virus. The effect of viable mutations on viral growth will be discussed. These data allow us to further our understanding of the function of the NA in influenza replication.