ABSTRACT From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo . To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.