Natural products from the plants of the family Annonaceae, collectively called Annonaceous acetogenins, are very potent inhibitors of the NADH-ubiquinone reductase (Complex I) activity of mammalian mitochondria. The properties of five of such acetogenins are compared with those of rotenone and piericidin, classical potent inhibitors of Complex I. Rolliniastatin-1 and rolliniastatin-2 are more powerful than piericidin in terms of both their inhibitory constant and the protein-dependence of their titre in bovine submitochondrial particles. These acetogenins could be considered therefore the most potent inhibitors of mammalian Complex I. Squamocin and otivarin also have an inhibitory constant lower than that of piericidin, but display a larger protein-dependence of the titre. Squamocin and otivarin, contrary to the other acetogenins, behave qualitatively like rotenone. Rolliniastatin-2 shows unique properties as its interaction, although mutually exclusive to that of piericidin, appears to be mutually non-exclusive to that of rotenone. It is the first time that a potent inhibitor of Complex I is found not to overlap the active site of rotenone.