The potential of wheat straw for ethanol production after pretreatment with O(3) generated in a plasma at atmospheric pressure and room temperature followed by fermentation was investigated. We found that cellulose and hemicellulose remained unaltered after ozonisation and a subsequent washing step, while lignin was degraded up to 95% by O(3). The loss of biomass after washing could be explained by the amount of lignin degraded. The washing water of pretreated samples (0-7 h) was analyzed for potential fermentation inhibitors. Approximately 30 lignin degradation products and a number of simple carboxylic acids and phenolic compounds were found, e.g., vanillic acid, acetic acid, and formic acid. Some components had the highest concentration at the beginning of the ozonisation process (0.5, 1 h), e.g., 4-hydroxybenzladehyde, while the concentration of others increased during the entire pretreatment (0-7 h), e.g., oxalic acid and acetovanillon. Interestingly, washing had no effect on the ethanol production with pretreatment times up to 1 h. Washing improved the glucose availability with pretreatment times of more than 2 h. One hour of ozonisation was found to be optimal for the use of washed and unwashed wheat straw for ethanol production (maximum ethanol yield, 52%). O(3) cost estimations were made for the production of ethanol at standard conditions.