Geographical spread of highly pathogenic avian H5N1 influenza viruses may give rise to an influenza pandemic. During the first months of a pandemic, control measures would rely mainly on antiviral drugs, such as the neuraminidase (NA) inhibitors oseltamivir and zanamivir. In this study, we compared the sensitivity to oseltamivir of the NA of several highly pathogenic H5N1 viruses isolated in Asia from 1997 to 2005. The corresponding IC50 values were determined using a standard in vitro NA-inhibition assay. The Km for the substrate and the affinity for the inhibitor (Ki) of the NA were determined for a 1997 and a 2005 virus, using an NA-inhibition assay on cells transiently expressing the viral enzyme. Our data show that sensitivity to oseltamivir of the NA of H5N1 viruses isolated in 2004 and 2005 is about 10-fold higher as compared to earlier H5N1 viruses or to currently circulating H1N1 viruses. Three-dimensional modelling of the N1 predicted that Glu248Gly and Tyr252His changes could account for increased sensitivity. Our data indicate that genetic variation in the absence of any drug selective pressure may result in significant variations of sensitivity to anti-NA drugs. Although the clinical relevance of a ten-fold increase in sensitivity of the NA to oseltamivir needs to be investigated further, the possibility that sensitivity to anti-NA drugs could increase (or possibly decrease) significantly even in the absence of treatment underline the need for a continuous evaluation of the impact of genetic drift on this parameter, especially for influenza viruses with pandemic potential.