The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to nickel (Ni) were investigated. Ni was very rapidly accumulated in the cells and the accumulation could be directly correlated with the increase of NiCl2 concentration in the medium. At 0.05 mM NiCl2 growth was stimulated, but at 0.5 mM NiCl2, the growth rate was reduced. An indication of alterations in the presence of reactive oxygen species was detected by an increase in lipid peroxidation at 0.5 mM NiCl2. Catalase (CAT; EC 188.8.131.52), glutathione reductase (GR; EC 184.108.40.206), ascorbate peroxidase (APX; EC 220.127.116.11), guaiacol peroxidase (GOPX; EC 18.104.22.168) and superoxide dismutase (SOD; EC 22.214.171.124) activities were increased, particularly at earlier NiCl2 exposure times and the activities were higher at 0.5 mM NiCl2 for most of exposure times tested. Non-denaturing PAGE revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differentially affected by NiCl2 treatment and one GR isoenzyme was increased by NiCl2. NiCl2 at 0.05 mM did not induce lipid peroxidation and the main response appeared to be via the induction of SOD, CAT, GOPX and APX activities for the removal of the reactive oxygen species and through the induction of GR to ensure the availability of reduced glutathione.