Spent mushroom substrates (SMS) from cultivation of shiitake (Lentinula edodes) on three hardwood species were investigated regarding their potential for cellulose saccharification and for ethanolic fermentation of the produced hydrolysates. High glucan digestibility was achieved during enzymatic saccharification of the SMSs, which was related to the low mass fractions of lignin and xylan, and it was neither affected by the relative content of lignin guaiacyl units nor the substrate crystallinity. The high nitrogen content in SMS hydrolysates, which was a consequence of the fungal pretreatment, was positive for the fermentation, and it ensured ethanol yields corresponding to 84–87% of the theoretical value in fermentations without nutrient ...