Calmidazolium (CMZ) is a positively charged, hydrophobic compound used as a calmodulin antagonist. It may cause unspecific effects in mitochondria, e.g., a decrease in membrane potential (ΔΨ), swelling, and uncoupling. Several groups have advised against use of CMZ in studying signal transduction in mitochondria. We report here that it interferes with measurement of ΔΨ in rat liver mitochondria (RLM) when using the tetraphenyl phosphonium (TPP + ) electrode. We also found that CMZ reduces the signal, indicating an apparent drop in ΔΨ. CMZ itself gave a signal with the TPP + electrode in the absence of RLM. At high concentrations, >10μM, it also reduced the fluorescence quenching of the probe rhodamine 123. This may be due to an interference with mitochondrial uptake and binding of this positively charged probe or to an uncoupling effect. It is concluded that CMZ and similar positively charged calmodulin antagonists such as trifluoperazine may be used in mitochondria if these interferences are controlled and calibration is carried out under the experimental conditions used.