The highly pathogenic avian influenza virus H5N1 is known to induce high level of tumor necrosis factor alpha (TNF-alpha) from primary macrophages. However, it is still unclear whether current H5N1 strains also induce high TNF-alpha production, as most of the data were derived from extinct clade 0 H5N1 strain. Here, we show that current clade 1 and 2 H5N1 strains induce variable levels of TNF-alpha that are not necessarily higher than those induced by seasonal influenza viruses. The result suggests that hyper-induction of TNF-alpha in human macrophages is not always associated with a highly pathogenic phenotype. We further tested the contribution of the NS gene segment from H5N1 isolates to TNF-alpha induction by using reverse genetics. While NS conferred some variation in TNF-alpha induction when incorporated into an H1N1 virus genetic background, it did not affect TNF-alpha induction in an H5N1 virus genetic background, suggesting that other viral genes are involved.