Three effects concerning the organic cations, the aggregation, the metachromasy and the pKα shift, have been studied in comparison at high dye concentrations, with natural and synthetic polyanions and with energized submitochondrial particles.The metachromatic effect has been obtained by increase of the free cationic dye concentration or by interaction of the dye with natural, synthetic polyanions and energized submitochondrial particles. The metachromatic effect is dependent on the pH of the medium, the presence of ionized acidic groups, and the ionic strength of the medium.The polyanion‐induced metachromasy requires the ionization of the acidic groups of the polyelectrolyte. The metachromasy with neutral red requires an acidic pH if induced by increase of the dye concentration or by chondroitin sulphuric acid, whereas it takes place also at alkaline pH if induced by polystyren sulphonic acid or by energized particles. It is inhibited by increase of ionic strength in the case of agar and chondroitin sulphuric acid, but not in the case of the energized particles or polystyrene sulphonic acid.Interaction of neutral red with polystyrene sulphonic acid or energized particles results also in a large apparent pKα shift, which represents the mechanism for obtaining the metachromatic effects at alkaline pH. The apparent pKα, as measured from the extinction of the alkaline band is: 6.0 at 400 μM neutral red, 6.7 at 20 μM neutral red, 7.0 with chondroitin sulphuric acid, 7.1 with the deenergized particles, 8.0 with polystyrene sulphonic acid and 8.1 with energized particles. From thermodynamic considerations it is suggested that the pKα shift requires a decrease of the activity coefficient of the dye following the formation of ion pairs with anionic groups of the membrane. The pKα shift may be taken as a tool for discriminating between a dyeinduced and a membrane‐induced metachromatic effect.A model is proposed for the energized membrane, based upon electrostatic and hydrophobic interactions of the cationic dyes with a layer of oriented nucleophilic sites in an environment of intermediate polarity.