Previous studies have indicated that semen of heat-stressed bulls shows impaired mitochondrial activity and high levels of oxidative stress, which may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components (Nichi et al. 2006 Theriogenology 66, 822–828). Disruption of the sperm mitochondria could have a potential damaging effect not only on an individual sperm cell but also on the surrounding cells, especially regarding the sperm membrane, possibly due to the release of a high amount of reactive oxygen species (ROS) produced in this environment (rich in electrons) that would then lead to oxidative stress. To test this hypothesis, semen samples of 11 Simmental bulls kept in tropical environments were collected during the summer months. Semen was evaluated as follows: the 3-3′ diaminobenzidine stain (DAB) as an index of mitochondrial activity, the hypo-osmotic swelling test (HOST) as an index of membrane integrity, measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation, and measurement of the enzymatic antioxidants superoxide dismutase, catalase and glutathione peroxidase activities. For correlation analysis, the Pearson test was used (variables were transformed when necessary), and for nonparametric variables, the Spearman rank test was used. A high positive correlation was found between sperm cells with highly active mitochondria (DAB class I) and the percentage of cells with intact membrane by HOST (r = 0.93; P < 0.05), and a negative correlation between the latter and the percentage of inactive mitochondria (r = –0.91; P < 0.05), indicating that the higher the percentage of cells showing impaired mitochondrial activity, the higher the percentage of cells with damaged membrane. There was also a positive correlation between TBARS and the percentage of cells with disrupted mitochondria (r = 0.86; P < 0.05), indicating that the higher the percentage of sperm with impaired mitochondrial activity, the higher the oxidative stress. No correlation existed between the enzymatic antioxidants and any of the variables studied. The results indicate that heat stress may lead to an increase in testicular ROS levels, overcoming the seminal antioxidant protection. This, in turn, may cause damage of the mitochondria and a subsequent release of more pro-oxidative substances, and an exponential increase of oxidative stress. Understanding these mechanisms may lead to more tailored antioxidant therapies in the future.