Abstract A wild type (WT) and a colourless mutant (CM) of Euglena gracilis were used to study the biochemical effects of ozone (O 3) stress on the components of the ascorbate-glutathione cycle (AA-GSH cycle). The WT cells had higher initial ascorbate (AA) and ascorbate peroxidase (APX, EC 184.108.40.206) activity but lower glutathione (GSH), glutathione reductase (GR, EC 220.127.116.11) and glucose-6-phosphate dehydrogenase (G6PDH, EC 18.104.22.168) activities than the CM cells. Ozone exposure (1.0 μmol O 3·min −1) of both cell types resulted in a viability loss of 9 % and 15 % for the WT and CM cells, respectively. After O 3 exposure, a similar reduction in GSH content was observed in both cell types, whereas the other components of the AA-GSH cycle showed no change. Our results indicate that the higher tolerance of the WT cells to O 3 is associated with a higher initial AA content. To confirm this, the endogenous AA content in both cell types was modified by adding L-galactono-γ-lactone (L-GAL) or D-galactono-γ-lactone (D-GAL) to the growth medium. L-GAL treatment resulted in a 5.2-fold increase in the initial AA content and an increase of O 3 tolerance in the CM cells. On the other hand, D-GAL treatment decreased the AA content as well as O 3 tolerance of WT cells. Together, these results suggest that O 3 tolerance is closely correlated with the AA content in Euglena cells.