The objectives were to determine factors affecting generation of reactive oxygen species (ROS) in porcine embryos after electrical activation of oocytes, and the effects of an antioxidant and chemical agent on ROS generation. Greater ROS were induced by electrical activation compared to IVF (mean+/-S.E.M., 14.6+/-0.8 vs. 9.2+/-0.4, P<0.05). Furthermore, ROS generation in embryos after electrical activation was significantly increased by higher intensity and longer duration electrical pulses and by higher exogenous Ca(2+) concentrations. Cleavage rate and blastocyst formation rate were not directly related to the level of ROS. Supplementation of the IVC medium with 0.5mM glutathione (GSH) reduced ROS (9.2+/-0.4 vs. 14.7+/-0.9, P<0.05). Treatment with the chemical activation agent, 6-dimethylaminopurine (6-DMAP) for 3h did not induce further ROS generation in combination with electrical activation, but it improved blastocyst formation rate (53.8+/-1.1 vs. 23.7+/-3.5, P<0.05). We concluded that generation of ROS should be considered for optimizing electrical activation and that supplementing an antioxidant or combining electrical and chemical activation induced lower ROS generation in electrically activated porcine embryos.