The relative stereochemistry between C(2) and C(4) of the naturally occurring, rearranged acetonylbutyrolactone-containing annonaceous acetogenins has been determined. Model lactones 4c, 4t, 6c, and 6t, which mimic the rearranged portion of the natural products in question, have been synthesized and unambiguously assigned as having cis or trans stereochemistry on the basis of 1H NMR NOE experiments. Each stereoisomeric pair displays diagnostic coupling constant and chemical shift trends. These data were then compared with those of all of the pertinent acetogenins bullatacinone (2), squamone (7), isoannonacin (8), isoannonacin-10-one (9), and rollinone (10) and some of their peracetate derivatives. For none of these compounds had the C(2)/C(4) relative stereochemistry been previously determined. The major (for 2 and 10) or only reported isomers (for 7-9) bear a trans C(2)/C(4) relationship.