Influenza A(H1N1) viruses resistant to oseltamivir carboxylate (OC) emerged in 2007/2008 in the absence of antiviral pressure. These OC-resistant A(H1N1) viruses had a better fitness than the sensitive ones as they were 100% prevalent in 2008/2009. To better understand the role of the neuraminidase (NA) affinity in the emergence of these OC-resistant A(H1N1) viruses we compared the NA properties among A(H1N1) clinical isolates in south of France between 2005 and 2009 and reference strains from 1977 to 2007, using NA inhibition assays, kinetic analyses of NA activities, and sequence analysis of viral NA and hemagglutinin (HA). In 2007/2008, among 374 A(H1N1) isolates tested, 38% were resistant to OC with a mean IC50 of 564+/-357 nM. The mean Km of OC-sensitive isolates (H275) was significantly lower (22.6+/- 4.7 microM) than the Km of previous reference strains (44.9+/- 5 microM) and the mean Km of the OC-resistant isolates (Y275) (37.2 +/- 7.7 microM). The combination of different amino acid mutations in N1 particularly the D344N could explain the higher NA affinity of A/Brisbane/59/2007 related variants compared to the previous A(H1N1) strains and the H275Y mutation allowed to retrieve Km values near 40 microM.