In conclusion, by comparing the ability of different H5-HA to produce pseudotyped particles, we have demonstrated that when a single A134V mutation is introduced in the receptor binding site, the ability of the usually inefficient H5Anh to generate H5pp is largely restored. It is likely that the A134V mutation leads to an increased level of cell surface HA expression and reduced binding to sialic acid receptors, both of which contribute to the production of H5pp. The A134V mutation has been reported as a naturally occurring mutation in human host; and importantly, this mutation is so far only found in human isolates of H5N1 viruses. Our data with hemagglutination assays further demonstrate that viral isolates from human cases with avian influenza carrying the A134V substitution exhibit a reduced binding to alpha-2,3 linked sialic acids. Therefore, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 viruses. It is possible that other mutations leading to reduction in receptor binding may exist and cause a change in pseudotyping efficiency. Thus, H5pp production together with soluble HA protein cell binding analysis may serve as convenient functional assays to screen for mutations with potential consequences on receptor binding properties and host adaptations of H5N1 viruses. Although zoonotic transmission from poultry to humans remains inefficient for H5N1, it may be of importance to monitor closely mutations in regions of the receptor binding site of H5-HA.