The function of microbial MDRs remains a hotly debated subject. Given the very broad substrate specificities of some MDRs, like the RND pumps that can extrude all classes of amphipathic compounds (cationic, neutral, and anionic), it seems difficult to develop a rationale for pinpointing possible natural substrates of these translocases. At the same time, several clues can be used to guide our search for natural MDR substrates. One is the fact that amphipathic cations appear to be the preferred substrates of MDRs. These substances are extruded by MDRs of all 5 known families and are the almost exclusive substrates of SMR and MF family MDRs. The universal nature of amphipathic cations as MDR substrates suggests that these were the substances that fueled the evolution of MDR pumps. Two factors apparently favored this particular class of molecules for the role of original MDR substrates--need and opportunity. Unlike other substances, amphipathic cations accumulate in the cell driven by the membrane potential, which makes cations potentially the most dangerous toxins. At the same time, amphipathic cations are highly hydrated and do not permeate the membrane as readily as neutral compounds, making it feasible to design a defense based on an efflux pump. The paucity of known cationic (non-basic) antimicrobials might be a result of using MDR-expressing microbial cells for antibiotic discovery. Plant amphipathic cations, the berberine alkaloids, are good MDR substrates. The Berberis plants produce 5'-methoxyhydnocarpin-D, an MDR inhibitor that potentiates the action of berberine. It is suggested that the further evolution of MDR pumps was determined largely by the barrier function of the membrane they reside in. Thus Gram negative bacteria have an outer membrane barrier that slows the penetration of virtually all amphipathic molecules, and transenvelope MDRs of the RND and EmrAB-type extrude their substrates across this barrier. A low permeability of the cytoplasmic membrane of yeast similarly allows for the operation of broad-specificity ABC and MF MDRs. The presence of MDR sensors that regulate the expression of some MDR pumps strongly suggests that defense against external toxins is the function of these MDRs. The BmrR transcriptional activator of the MerR family induces expression of the Bmr pump in B. subtilis and is a sensor specifically designed to recognize amphipathic cations. Similarly, the OacR repressor binds chemically unrelated cations, which leads to the expression of the QacA pump in S. aureus. In E. coli, the EmrR sensor of the MarR repressor family binds unrelated neutral molecules, allowing for expression of the transenvelope EmrAB pump.