This study shows that high pathogenic H5N1 influenza virus infection of chicken induced high levels of bioactive interferon type I in the lung (4.3 × 10(5) U/mg tissue), plasma (1.1 × 10(5) U/mL), and spleen (9.1 × 10(5) U/mg tissue). In contrast, a low pathogenic attenuated H5N1 vaccine strain only induced approximately 24 times less IFN in the lung, 441 times less in the spleen and 649 less in the plasma. This was in the same range as a reassortant carrying the HA from the vaccine strain and the remaining genes from the high pathogenic virus. On the other hand, a reassortant virus with the HA from the high pathogenic H5N1 with the remaining genes from the vaccine strain had intermediate levels of IFN. The level of interferon responses related to the viral load, and those in the spleen and blood to the spread of virus to lymphoid tissue, as well as disease severity. In vitro, the viruses did not induce interferon in chicken embryonic fibroblasts, but high levels in splenocytes, with not clear relationship to pathogenicity and virulence. This, and the responses also with inactivated viruses imply the presence of plasmacytoid dendritic cell-like leukocytes within the chicken immune system, possibly responsible for the high interferon responses during H5N1 infection. Our data also indicate that the viral load as well as the cleavability of the HA enabling systemic spread of the virus are two major factors controlling systemic IFN responses in chicken.