The neuraminidase (NA) of influenza viruses cleaves sialic acids from receptors, prevents self-aggregation and facilitates release of virus during budding from host cells. Although the structure and function of the globular head of the influenza virus NA has been well studied, much less is known about the stalk of the NA, the region between the viral membrane and the globular head. Applying a reverse genetics system, we altered the stalk of the influenza A/WSN/33 virus NA by making deletions, insertions and mutations in this region of the gene. Our data show that the length of the NA stalk can be variable. Deletions of up to 28 amino acids and insertions of up to 41 amino acids in the stalk region did not abolish formation of infectious progeny virus. The data also indicate that the cysteine at position 76 is essential for formation of infectious virus, and that deletions beyond the cysteine did not result in infectious virus. Interestingly, shortening of the length of the stalk region by 28 amino acids resulted in a virus with a markedly reduced growth rate in MDCK cells as compared to that in MDBK cells. An insertion of 41 extra amino acids into the stalk did not significantly interfere with viral growth in MDCK or MDBK cells, which suggests that the stalk region would tolerate the introduction of long foreign sequences.