The cationic fluorescent dyes, berberines, have been observed to inhibit NAD-linked respiration in rat liver mitochondria. Low concentrations inhibit electron transport in the NAD-ubiquinone span after penetration into mitochondria. More hydrophobic alkyl derivatives proved to be stronger inhibitors showing more rapid onset of inhibition. The inhibition was totally dependent on the energization of the membrane; however, the addition of a hydrophobic anion stimulated the inhibition effects in uncoupled mitochondria. Substantially higher concentrations of berberines are needed for the inhibition of the oxidation of succinate. The excess of dye interacting with surface dipoles in the energized state can inhibit the energy transduction through the complexbc ₁. On the basis of the difference in the rate of fluorescence response when berberines are added to coupled mitochondria and the corresponding inhibition effects, the presence minimally of two binding sites was suggested. The dye bound on the outer surface is highly fluorescent and inhibits the energy transduction if added in excess. The remaining dye interacting with NADH dehydrogenase does not fluoresce. The accumulation of alkylberberine in mitochondria results in additional effects in the region of cytochromeb the nature of which is not fully understood.