Stereospecific H-1 and C-13 resonance assignments of hexadienoyl-CoA and crotonyl-CoA have been determined. The two diastereotopic methyl groups at the C2 " carbon were assigned via transferred nuclear Overhauser effect experiments on the complex of hexadienoyl-CoA with the enzyme enoyl-CaA hydratase. The two diastereotopic 1 " protons were assigned using heteronuclear multiple-bend correlation experiments in conjunction with rotating frame nuclear Overhauser spectroscopy. This represents the first set of complete stereospecific assignments for any CoA derivative. The assignments allow a detailed quantitative conformational analysis of the uncomplexed form of the molecule. In particular, the relative population of the various rotamers about the C1 "-C2 " and the C2 "-C3 " bonds have been determined. The database of protein-bound CoA structures has been surveyed and used to compare the structure(s) of CoA in protein-CoA complexes with the conformational preferences of free CoA.