The relationship between the protonation state of ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), an antitumoral alkaloid, and the transmembrane proton electrochemical gradient associated with mitochondrial membranes has been investigated. The binding parameters of ellipticine to mitochondria are independent of either the transmembrane Delta pH or membrane potential, which are both components of proton gradient (association constant = 1.5 X 10(6) M(-1); maximal binding ratio = one bound ellipticine per 25 phospholipids). Since the apparent pK of bound ellipticine is 7.1, its two protonation states can be detected from their specific fluorescence emission spectra near physiological pH. It is shown that a shift in the drug protonation equilibrium toward the neutral form occurs in mitochondrial membrane during the generation of the proton electrochemical gradient. This shift is sensitive to transmembrane Delta pH but insensitive to membrane potential, indicting that the protonation equilibrium of membrane bound ellipticine detects the H+ movement in mitochondria.