The aim of this study was to assess combined effect of 1 kJm -2 d -1UV-B radiation and substrate acidity (pH 4.8) on photosynthesis and growth of summer rape (Brassica napus) in a controlled environment. Rape were sown in a neutral (pH 6.0-6.5) peat substrate, when 2nd true leaf unfolded, growth substrate was acidified using 0.07 mM H 2 SO 4 concentration solution and UV-B radiation exposure was started. Duration of the experiment: five days. Gas exchange parameters were measured with portable photosynthesis system LI-6400. Content of chlorophyll was evaluated spectrophotometrically. The results showed that the highest inhibition on Brassica napus growth and photosynthetic parameters was under combined effect of 1 kJm -2 d -1 UV-B radiation and pH 4.8 substrate acidity. The impact of single 1 kJm -2 d -1 UV-B radiation on investigated parameters was low and statistically insignificant, except transpiration rate and water use efficiency. Single substrate acidity affected in 58.6% (p < 0.05) decrease in photosynthetic rate, almost twofold decrease of transpiration rate, and 3.8% (p < 0.05) decrease in intercellular CO 2 concentration, and 27.1% (p < 0.05) increase in water use efficiency, compared to the reference treatment. Combined substrate acidity and UV-B radiation effect on photosynthetic rate was the highest, when the decrease was 80.6% (p < 0.05), compared to the reference treatment. The changes of transpiration rate under combined effect of investigated stressors compared to the reference treatment were insignificant (p > 0.05). The increase (by 2%, p < 0.05) of intercellular CO 2 concentration of Brassica napus affected by combined impact of investigated stressors shows that higher negative impact was on enzymatic reactions of photosynthesis than on transpiration. The highest negative effect on content of photosynthetic pigments and biomass accumulation also was of combined effect of investigated stressors.