Technical Abstract: Since 2002, H5N1 highly pathogenic avian influenza (HPAI) viruses have caused mortality in numerous species of wild birds; this is atypical for avian influenza virus (AIV) infections in wild birds, especially for species in the Order Anseriformes. Although these infections document the susceptibility of wild birds to H5N1 HPAI viruses and the spillover of these viruses from infected domestic birds to wild birds, it is unknown if H5N1 HPAI viruses can persist in free-living avian populations. In a previous study, we established that wood ducks (Aix sponsa) are highly susceptible to infection with H5N1 HPAI viruses and shed moderately high concentrations of virus for several days. In order to quantify this susceptibility and further evaluate the likelihood of H5N1 HPAI viral maintenance in a wild bird population, we determined the minimal concentration of virus required to produce infection in wood ducks. To accomplish this, 25 3- to 4-mo-old wood ducks were inoculated intranasally with decreasing concentrations of a H5N1 HPAI virus (A/Whooper Swan/Mongolia/244/05 [H5N1]). The minimal infectious dose and lethal dose of H5N1 HPAI virus in wood ducks were very low (0.75log10 and 1.35log10 50% mean tissue culture infectious dose (TCID50)/ml, respectively) and less than that of chickens (2.8log10 and 2.8log10 TCID50/ml). These results confirm that wood ducks are highly susceptible to infection with H5N1 HPAI virus. The data from this study, combined with what is known experimentally about H5N1 HPAI virus infection in wood ducks and viral persistence in aquatic environments, suggest that the wood duck would represent a sensitive indicator species for H5N1 HPAI. Results also suggest that the potential for decreased transmission efficiency associated with reduced viral shedding (especially from the cloaca) and a loss of environmental fitness (in water), may be offset by the ability of this virus to be transmitted through a very low infectious dose.