In isolated rat hepatocytes a sustained high increase in intracellular free Ca2+ ([Ca2+]i), induced by extracellular ATP, is associated with mitochondrial dysfunction and cell death. The Ca(2+)-induced effects are Pi-dependent and less severe when the intracellular K+ content is low. In this study, the involvement of mitochondrial K+ processing in Ca(2+)-induced loss of mitochondrial membrane potential (MMP) and viability was investigated. The recently introduced K(+)-sensitive dye PBFI (K(+)-binding benzofuran isophthalate) has been used in combination with video-microscopy to assess intramitochondrial free K+ concentration ([K+]mito) in rat liver mitochondria in situ. After rapid permeabilization of the plasma membrane to remove cytosolic PBFI, the remaining PBFI was localized in mitochondria, and a 'resting' [K+]mito of approx. 15 mM could be measured. Increased [K+]mito levels were measured after induction of a prolonged increase in [Ca2+]i by ATP. Much lower [K+]mito, more comparable with control levels, were observed when intracellular K+ was depleted by omission of extracellular K+. In permeabilized cells the Ca(2+)-induced, Pi-dependent, dissipation of the MMP was markedly delayed in the absence of K+. These observations suggest involvement of [K+]mito as modulating agent in Ca(2+)-induced cytotoxicity in hepatocytes.