Characterizing a stereoisomer library of 28 of the 64 possible isomers of the acetogenin murisolin, including 24 of the 32 possible diastereomers, provides a complete picture of the spectra of this class of molecules. Remarkably, each of the 32 diastereomers exhibits one of only six sets of substantially identical (1)H NMR spectra under standard conditions. These spectra follow directly from a local symmetry analysis of the dihydroxy-THF fragment of the molecule and provide no information about the configuration about the hydroxybutenolide. Eighteen tris-Mosher ester derivatives of library members have been made, and their spectra were analyzed to give a complete picture of the usefulness of chiral derivatives. The tris-Mosher esters of the 64 isomers of murisolin will exhibit 40 sets of spectra: 16 isomers have unique spectra whereas 24 isomers share an identical spectrum with one other isomer. This identity occurs even though the pairs of compounds were already diastereomers (not enantiomers) before the derivatization. The complete set of spectra allows any murisolin or closely related compound to be narrowed to one or two structures by simple matching and without recourse to assignment and subtraction of resonances. The structure of murisolin was proved to be the 4R,15R,16R,19R,20R,34S isomer, whereas the assignment of 16,19-cis-murisolin as RRRSSS was changed to the RSSRRS diastereomer and murisolin A is suggested to be RSRRRS.