Japanese cedar (Cryptomeria japonica) shares around 60% of plantation forests in Japan, and there is a growing demand for thinning of the forest. However, the softwood is one of the most recalcitrant wood species for hydrothermal and thermochemical pretreatments for enzymatic saccharification. In the present paper, we applied combined pretreatments by solvolysis and cultivation with white rot fungi to develop environmentally benign pretreatment system applicable to recalcitrant softwood. Due to the recalcitrance of the softwood, enzymatic saccharification yield from ethanolysis pulp was 10.2%, based on the weight of holocellulose. To increase the sugar yield, the softwood was treated with selective white rot fungi prior to the ethanolysis. Treatment of the softwood with a biopulping fungus, Ceriporiopsis subvermispora FP-90031 and a new fungal isolate Phellinus sp. SKM2102 for 8 weeks increased the sugar yield to 35.7 and 40.8%, respectively. The best pretreatment conditions in terms of overall sugar yield including a soluble fraction were obtained by ethanolysis after the fugal treatment with Phellinus sp. SKM2102, resulting in production of 42.2 g of total reducing sugars per 100 g of the fungus-pretreated biomass. After the combined pretreatment, simultaneous saccharification and fermentation of the water-insoluble pulp fraction were carried out using Saccharomyces cerevisiae. Ethanol production from undecayed Japanese cedar wood was negligible but pretreatments with the two fungi significantly increased the ethanol production, in combination with ethanolysis. The combined pretreatment with solvolysis and Phellinus sp. SKM2102 is attractive for biorefinery of the recalcitrant softwood. (C) 2010 Elsevier Ltd. All rights reserved.