The effect of different ozonation conditions on straw from Secale cereale (rye straw) pretreatment has been investigated. Using the Taguchi method, this study analyzed the optimum conditions for pretreatment of rye straw by ozonation. After 60 min of rye straw ozonation the concentration of reducing sugars (RS) and volatile fatty acid (VFA), chemical oxygen demand (COD) were 7.4, 32.3 and 11.7 times higher, respectively compared to samples raw rye straw. The most effective rye straw ozonation occurred while using the highest amount of the rye straw (15 g) treated with lower ozone dose (100 gO(3)/m(3)) in the longest period of time (60 min). For this variant of experiment the increment of methane production was 291.71 dm(3) CH4/kg VS. Moreover, co-digestion of sewage sludge with addition of 20% ozonated rye straw allowed to obtain 269.1 dm(3) CH4/kg VS. The positive effect of ozone on changes in the rye straw structure has been confirmed by SEM and FTIR analysis.