The opening of the permeability transition pore (PTP) in mitochondria is a key event in the initiation of cell death in various pathologic states, including ischemia/reperfusion. The activation of K transport into mitochondria protects cells from ischemia/reperfusion. However, the role of K transport in PTP regulation is unclear. Here, we studied the role of K and other monovalent cations in the regulation of the PTP opening in an in vitro model. The registration of the PTP opening, membrane potential, Ca-retention capacity, matrix pH, and K transport was performed using standard spectral and electrode techniques. We found that the presence of all cations tested in the medium (K, Na, choline, and Li) strongly stimulated the PTP opening compared with sucrose. Several possible reasons for this were examined: the effect of ionic strength, the influx of cations through selective and non-selective channels and exchangers, the suppression of Ca/H exchange, and the influx of anions. The data obtained indicate that the mechanism of PTP stimulation by cations includes the suppression of K/H exchange and acidification of the matrix, which facilitates the influx of phosphate. Thus, the K/H exchanger and the phosphate carrier together with selective K channels compose a PTP regulatory triad, which might operate in vivo.