BACKGROUND: Influenza viruses attach to cells via sialic acid receptors. The viral neuraminidase (NA) is needed to remove sialic acids so that newly budded virions can disperse. Known mechanisms of resistance to NA inhibitors include mutations in the inhibitor binding site, or mutations in the hemagglutinin that reduce avidity for sialic acid and therefore reduce the requirement for NA activity. RESULTS: Influenza H3N2 isolates A/Oklahoma/323/03 (Fujian-like), A/Oklahoma/1992/05 (California-like), and A/Oklahoma/309/06 (Wisconsin-like) lost NA activity on passage in MDCK cells due to internal deletions in the NA-coding RNA segment. The viruses grow efficiently in MDCK cells despite diminished NA activity. The full length NA enzyme activity is sensitive to oseltamivir but replication of A/Oklahoma/323/03 and A/Oklahoma/309/06 in MDCK cells was resistant to this inhibitor, indicating that NA is not essential for replication. There was no change in HA activity or sequence after the NA activity was lost but the three viruses show distinct, quite restricted patterns of receptor specificity by Glycan Array analysis. Extensive predicted secondary structure in RNA segment 6 that codes for NA suggests the deletions are generated by polymerase skipping over base-paired stem regions. In general the NA deletions were not carried into subsequent passages, and we were unable to plaque-purify virus with a deleted NA RNA segment. CONCLUSION: H3N2 viruses from 2003 to the present have reduced requirement for NA when passaged in MDCK cells and are resistant to NA inhibitors, possibly by a novel mechanism of narrow receptor specificity such that virus particles do not self-aggregate. These viruses delete internal regions of the NA RNA during passage and are resistant to oseltamivir. However, deletions are independently generated at each passage, suggesting that virus with a full length NA RNA segment initiates the first round of infection.