Electrophysiological techniques have been integral to our understanding of protein translocation across various membranes, and, in particular, the mitochondrial inner and outer membranes. Descriptions of various methodologies (for example, patch clamp, planar bilayers, and tip dip, and their past and potential contributions) are detailed within. The activity of protein import channels of native mitochondrial inner and outer membranes can be studied by directly patch clamping mitochondria and mitoplasts (mitochondria stripped of their outer membrane by French pressing) from various genetically manipulated strains of yeast and mammalian tissue cultured cells. The channel activities of TOM, TIM23, and TIM22 complexes are compared with those reconstituted in proteoliposomes and with those of the recombinant proteins Tom40p, Tim23p, and Tim22p, which play major roles in protein translocation. Studies of the mechanism(s) and the role of channels in protein translocation in mitochondria are prototypes, as the same principles are likely followed in all biological membranes including the endoplasmic reticulum and chloroplasts. The ability to apply electrophysiological techniques to these channels is now allowing investigations into the role of mitochondria in diverse fields such as neurotransmitter release, long-term potentiation, and apoptosis.