Wheat straw was subjected to four steam treatments with addition of 1.8% sulphuric acid (dry matter (DM) basis) at various temperatures (room temperature (control), 98, 116 and 134°C) for 120 min. The effect of drying post-treatment was investigated by dividing washed steam-treated samples into two groups — undried and dried (oven dried at 105°C overnight). Samples were analysed for carbohydrate composition (neutral sugars), hydrolysed hemicellulose, and total and soluble phenolic content. Sample pore size distribution was estimated by the accessible pore volume (APV) at various probe diameters (fructose, raffinose and eight dextrans with different molecular diameter). Biological availability of the cell wall fraction was measured by in vitro gas production (rumen microbes) and enzymic hydrolysis (free polysaccharidases). Increasing temperatures of treatment (control, 98, 116 and 134°C) resulted in increasing solubility of the hemicellulose (0.0%, 5.8%, 20.4% and 50.3%, respectively) and soluble phenolics (2.2%, 2.4%, 2.8% and 3.2%, respectively). APV measured at various molecular diameters was also increased as a result of steam treatment. However, no significant changes were found in APV within steam-treated samples. Biological assays showed that rate of gas production by rumen microbes (3.2, 3.2, 3.6 and 4.0% h −1) or extent of enzymic hydrolysis at 24 h (91.6, 110.5, 147.8 and 173.7 mg total sugar g −1 substrate) were significantly increased at higher temperatures. Drying post-treatment had a negative effect on the utilization of steam-treated samples measured either by gas production or enzymic hydrolysis. This negative effect corresponds to a lower APV as measured at various molecular diameters after drying. Steam-treated samples had the following APV measured at 4.9 nm before and after drying, respectively: 0.25 ml g −1 and 0.15 ml g −1 at 98°C, 0.19 and 0.13 ml g −1 at 116°C, and 0.29 ml g −1 and 0.10 ml g −1 at 134°C. These results suggest that the positive effect of steam treatment conducted at temperatures lower than 134°C are mainly due to partial hemicellulose hydrolysis, lignin depolymerization and cell wall swelling. Furthermore, the collapse of the cell wall microstructure observed after oven drying can depress the utilization of steam-treated wheat straw either by rumen microbes or free enzymes.