The ability to synthesize particular steviol glycosides (SvGls) was studied inStevia rebaudianaBertoni hairy roots (HR) grown in the light or in the dark under the influence of different osmotic active compounds. Manipulation of culture conditions led to changes in the morphology and growth rate of HR, as well as to an increase in oxidative stress manifested as an enhancement in endogenous hydrogen peroxide concentration in the cultured samples. The highest level of H(2)O(2)was noted in HR cultured under light or in the medium with the highest osmotic potential. This correlated with the highest increase in the expression level ofent-kaurenoic acid hydroxylase, responsible for the redirection of metabolic route to SvGls biosynthesis pathway. An analysis of transcriptional activity of someUDPglucosyltransferase(UGT85c2,UGT74g1,UGT76g1) revealed that all of them were upregulated due to the manipulation of culture conditions. However, the level of their upregulation depended on the type of stress factor used in our experiment. Analysis of SvGls content revealed that HR grown under all applied conditions were able to synthesize and accumulate several SvGls but their concentration differed between the samples across the different conditions. The level of rebaudioside A concentration exceeded the content of stevioside in HR in all tested conditions. Concomitantly, the presence of some minor SvGls, such as steviolbioside and rebaudioside F, was confirmed only in HR cultured in the lowest osmotic potential of the medium while rebaudioside D was also detected in the samples cultured in the media supplemented with NaCl or PEG.