Using lignocellulosic biomass as an alternative resource for transportation fuel is an attractive prospect due to its abundance and low cost. Conventional pre-treatment methods such as steam explosion or ammonia fibre expansion need technologically complex equipment and high energy input, and they use toxic chemicals in order to achieve high glucose and ethanol yields. In this paper, the freezing pretreatment of barley straw is investigated as a low energy input and cost-effective alternative pretreatment method for second generation bioethanol production. In the freezing pre-treatment method, milled biomass mixed with water was frozen in temperatures as low as - 18 degrees C for a certain period of time and was then thawed to room temperature (around 22 degrees C). The freezing cycles were repeated several times so that the effect of repeated freezing could be studied. In addition, field experiments were carried out by taking samples of barley straw which had been stored outside, both in bales and swathes during a period of time between September and March in two consecutive years (in winter 2014-2015 and 2015-2016). Freezing pre-treatment was followed by enzymatic hydrolysis and fermentation. Glucose and ethanol yields and, additionally, hydrolysis and fermentation efficiencies were used as indicators of pre-treatment efficiency. The highest hydrolysis efficiency figure was 19.42%, which was achieved in laboratory tests where the biomass was frozen and thawed a total of four times. The best result from a field test was 10.28% from straw which had been stored in a swathe and which has been gathered in March. Fermentation yields ranged up to 88.80 g per kilogramme of biomass. Field tests showed that in the bale the temperature never fell below freezing and therefore the pre-treatment was not effective. When in the swathe the straw does freeze through, but the winter in Estonia was too changeable for this method to work effectively. (C) 2017 Elsevier Ltd. All rights reserved.