Nonesterified long-chain fatty acids (myristic, palmitic, oleic and arachidonic), added at low amounts (around 20 nmol/mg protein) to rat liver mitochondria, energized by respiratory substrates and suspended in isotonic solutions of KCl, NaCl, RbCl or CsCl, adjusted to pH 8.0, induce a large-scale swelling followed by a spontaneous contraction. Such swelling does not occur in alkaline solutions of choline chloride or potassium gluconate or sucrose. These changes in the matrix volume reflect a net uptake, followed by net extrusion, of KCl (or another alkali metal chloride) and are characterized by the following features: (1) Lowering of medium pH from 8.0 to 7.2 results in a disappearance of the swelling-contraction reaction. (2) The contraction phase disappears when the respiration is blocked by antimycin A. (3) Quinine, an inhibitor of the K(+)/H(+) antiporter, does not affect swelling but suppresses the contraction phase. (4) The swelling phase is accompanied by a decrease of the transmembrane potential and an increase of respiration, whereas the contraction is followed by an increase of the membrane potential and a decrease of oxygen uptake. (5) Nigericin, a catalyst of the K(+)/H(+) exchange, prevents or partly reverses the swelling and partly restores the depressed membrane potential. These results indicate that long-chain fatty acids activate in liver mitochondria suspended in alkaline saline media the uniporter of monovalent alkali metal cations, the K(+)/H(+) antiporter and the inner membrane anion channel. These effects are presumably related to depletion of mitochondrial Mg(2+), as reported previously [Arch. Biochem. Biophys. 403 (2002) 16], and are responsible for the energy-dissipating K(+) cycling. The uniporter and the K(+)/H(+) antiporter are in different ways activated by membrane stretching and/or unfolding, resulting in swelling followed by contraction.