Light-induced proton uptake by spinach chloroplasts is enhanced several-fold by 9-(4-diethylamino-1-methylbutylamino)-6-chloro-2-methoxyacridine (atebrin). This stimulation does not depend on the chlorophyll concentration. The amount of extra protons taken up in the presence of atebrin is determined by the pKa values of atebrin and the pH of the incubation medium. 2. Both the stimulation of the proton uptake and the maximal binding capacity for atebrin is sensitive to uncouplers. However, the ratio of bound to free atebrin does not depend on the presence of uncoupler up to the saturating atebrin concentration. 3. From simultanious kinetic measurements of atebrin fluorescence and proton movement it seems that after binding of the completely protonated atebrin the dye and the protons can move separately. This can also be inferred from the spectral behaviour of atebrin in illuminated chloroplasts. 4. The stimulation of the proton uptake by atebrin does not depend on the presence of salts in the incubation medium. However, the 'saturating' atebrin concentration increases strongly with increasing salt concentration in the medium. 5. It is concluded that the interaction of atebrin and other acridines with energized chloroplasts most likely occurs at the level of the membrane proper. 6. It is proposed that uncoupling by atebrin is a consequence of the creation of a high proton activity at the periphery of the thylakoid membrane, which opposes a proton gradient across the membrane. The uncoupling by atebrin is not of the protonophoric type according to this mechanism.
