For most cell types, fatty acids are excellent respiratory substrates. After being transported across the outer and inner mitochondrial membranes they undergo beta-oxidation in the matrix and feed electrons into the mitochondrial energy-conserving respiratory chain. On the other hand, fatty acids also physically interact with mitochondrial membranes, and possess the potential to alter their permeability. This occurs according to two mechanisms: an increase in proton conductance of the inner mitochondrial membrane and the opening of the permeability transition pore, an inner membrane high-conductance channel that may be involved in the release of apoptogenic proteins into the cytosol. This article addresses in some detail the mechanisms through which fatty acids exert their protonophoric action and how they modulate the permeability transition pore and discusses the cellular effects of fatty acids, with specific emphasis on their role as potential mitochondrial mediators of apoptotic signaling.