The effect of free fatty acids (FFAs) and acyl-CoA esters on K(+) uptake was studied in mitochondria isolated from durum wheat (Triticum durum Desf.), a species that has adapted well to the semi-arid Mediterranean area and possessing a highly active mitochondrial ATP-sensitive K(+) channel (PmitoK(ATP)), that may confer resistance to environmental stresses. This was made by swelling experiments in KCl solution under experimental conditions in which PmitoK(ATP) activity was monitored. Linoleate and other FFAs (laurate, palmitate, stearate, palmitoleate, oleate, arachidonate, and the non-physiological 1-undecanesulphonate and 5-phenylvalerate), used at a concentration (10 μM) unable to damage membranes of isolated mitochondria, stimulated K(+) uptake by about 2-4-fold. Acyl-CoAs also promoted K(+) transport to a much larger extent with respect to FFAs (about 5-12-fold). In a different experimental system based on safranin O fluorescence measurements, the dissipation of electrical membrane potential induced by K(+) uptake via PmitoK(ATP) was found to increase in the presence of 5-phenylvalerate and palmitoyl-CoA, both unable to elicit the activity of the Plant Uncoupling Protein. This result suggests a direct activation of PmitoK(ATP). Stimulation of K(+) transport by FFAs/acyl-CoAs resulted in a widespread phenomenon in plant mitochondria from different mono/dicotyledonous species (bread wheat, barley, triticale, maize, lentil, pea, and topinambur) and from different organs (root, tuber, leaf, and shoot). Finally, an increase in mitochondrial FFAs up to a content of 50 nmol mg(-1) protein, which was able to activate PmitoK(ATP) strongly, was observed under hyperosmotic stress conditions. Since PmitoK(ATP) may act against environmental/oxidative stress, its activation by FFAs/acyl-CoAs is proposed to represent a physiological defence mechanism.