The 3''-hydroxyl group of the pantetheine moiety of coenzyme-A generates diastereotopic methyl groups and protons at the 2''- and 1''-carbons, respectively. An analysis of the available crystal structures of coenzyme-if complexed at enzyme active sites suggests a common conformation for the pantoic acid portion of pantetheine. In this conformation, the preferred conformations about the O(1'')-C1'' bond is anti, about the C1''-C2'' bond gauche, and about the C2''-C3'' anti. The reported 1 H- 1 H nuclear Overhauser enhancements between these protons are consistent with the observed crystal structure conformations and facilitate the assignments made to the diastereotopic resonances. A HeteroCOSY spectrum allowed an unequivocal and complete assignment of the 1 3 C NMR for coenzyme-A, resolving the discrepancies between the assignments made by Roeder et al. (Physiol. Chem. Phys.7, 115-122 (1975)) and Patel and Walt (Anal. Biochem.170, 355-360 (1988)).