Barley was harvested at moisture contents of 36, 25 and 23% and infected with storage-associated moulds. Part of each lot of barley was also treated with lactobacilli or lactobacilli and yeast before being stored in airtight pilot silos. During storage the contents of starch, crude protein and β-glucans in the nine lots of barley remained fairly constant while the solubility of these components changed, especially in the barleys with 36% moisture. Treatment of the barleys with protecting microorganisms did not, to any large extent, influence the content or solubility of crude protein, starch or mixed-linked β-glucans during storage. Barley harvested at 36% moisture had higher extract viscosity and a higher dry matter disappearance in vitro than barley harvested at 23% moisture. Airtight storage diminished extract viscosity but had little effect on dry matter disappearance in vitro. Live weight of chickens at Days 14 and 21 was not significantly influenced by type of storage (dry or airtight) or microbial protection of the airtight stored 25% moisture barley. β-Glucanase supplementation significantly increased live weight at both Days 14 and 21. Feed conversion efficiency with airtight-stored high-moisture barleys was significantly improved by β-glucanase supplementation.