The yeast uses fermentation as the preferred pathway to obtain ATP and requires the respiratory chain to re-oxidize the NADH needed for activity of Glyceraldehyde-3-phosphate. This process is favored by uncoupling of oxidative phosphorylation (OxPhos), which is at least partially controlled by the mitochondrial unspecific pore (MUC). When mitochondrial ATP synthesis is needed as in the diauxic phase or during mating, a large rise in Ca concentration ([Ca]) closes MUC, coupling OxPhos. In addition, MUC opening/closing is mediated by the ATP/ADP ratio, which indicates cellular energy needs. Here, opening and closing of MUC was evaluated in isolated mitochondria from at different incubation times and in the presence of different ATP/ADP ratios or varying [Ca]. Measurements of the rate of O consumption, mitochondrial swelling, transmembrane potential and ROS generation were conducted. It was observed that MUC opening was reversible, a high ATP/ADP ratio promoted opening and [Ca] closed MUC even after several minutes of incubation in the open state. In the absence of ATP synthesis, closure of MUC resulted in an increase in ROS.