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, Ca2+-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 Ca2+/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.