The effect of spermine on Ca2+-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,ω-hexadecanedioic acid was studied. It has been shown that spermine inhibited the cyclosporin A-insensitive mitochondrial swelling induced by palmitic acid and Ca2+ and α,ω-hexadecanedioic acid and Ca2+. 100 μM spermine did not influence the mitochondrial respiration in state V2 and the respiration stimulated by palmitic acid, α,ω-hexadecanedioic acid and Ca2+. Preincubation of liposomes with 100 μM spermine resulted in inhibition of the palmitic acid/Ca2+- and α,ω-hexadecanedioic acid/Ca2+-induced release of a fluorescent dye sulforhodamine B from liposomes. At the same time, spermine added to fatty acid-containing liposomes stimulated Ca2+-dependent release of sulforhodamine B from liposomes. Addition of spermine to liposomes resulted in a significant increase in the ζ -potential of liposomal membranes (from −39.8 to −18.6 mV). A possible mechanism of spermine influence on palmitic acid/Ca2+- and α,ω-hexadecanedioic acid/Ca2+-induced permeability transition in mitochondria and liposomes is discussed.