The Annonaceous acetogenins represent a class of compounds with diverse bioactivities, including promising cytotoxicites. These are due, at least in part, to inhibition of complex I in the oxidative phosphorylation pathway in mitochondria. Fourteen Annonaceous acetogenins were tested in a rat liver mitochondrial oxygen uptake assay to probe additional structure-activity relationships. In this subcellular assay, the activity of non-adjacent bis-THF ring acetogenins depends on the distance between the two THF rings; the activity decreases to that of a mono-THF ring acetogenin if the distance is too long. When one THF ring is replaced with a tetrahydropyran ring, the activity remains comparable. The configuration of the THF ring, in mono ring compounds, seems to be more important than stereochemical differences in the rings of adjacent bis-THF ring compounds. Bullatacin, an adjacent bis-THF ring acetogenin, was used as a standard compound in every run to normalize the data.